Evidence for a non-replicative intracellular stage of nontypable Haemophilus influenzae in epithelial cells

Nontypable Haemophilus influenzae (NTHi) is a Gram-negative, non-capsulated human bacterial pathogen, a major cause of a repertoire of respiratory infections, and intimately associated with persistent lung bacterial colonization in patients suffering from chronic obstructive pulmonary disease (COPD). Despite its medical relevance, relatively little is known about its mechanisms of pathogenicity. In this study, we found that NTHi invades the airway epithelium by a distinct mechanism, requiring microtubule assembly, lipid rafts integrity, and activation of phosphatidylinositol 3-kinase (PI3K) signalling. We found that the majority of intracellular bacteria are located inside an acidic subcellular compartment, in a metabolically active and non-proliferative state. This NTHi-containing vacuole (NTHi-CV) is endowed with late endosome features, co-localizing with LysoTracker, lamp-1, lamp-2, CD63 and Rab7. The NTHi-CV does not acquire Golgi- or autophagy-related markers. These observations were extended to immortalized and primary human airway epithelial cells. By using NTHi clinical isolates expressing different amounts of phosphocholine (PCho), a major modification of NTHi lipooligosaccharide, on their surfaces, and an isogenic lic1BC mutant strain lacking PCho, we showed that PCho is not responsible for NTHi intracellular location. In sum, this study indicates that NTHi can survive inside airway epithelial cells.

Characterization of the six glycosyltransferases involved in the biosynthesis of Yersinia enterocolitica serotype O:3 lipopolysaccharide outer core

Yersinia enterocolitica (Ye) is a gram-negative bacterium; Ye serotype O:3 expresses lipopolysaccharide (LPS) with a hexasaccharide branch known as the outer core (OC). The OC is important for the resistance of the bacterium to cationic antimicrobial peptides and also functions as a receptor for bacteriophage phiR1-37 and enterocoliticin. The biosynthesis of the OC hexasaccharide is directed by the OC gene cluster that contains nine genes (wzx, wbcKLMNOPQ, and gne). In this study, we inactivated the six OC genes predicted to encode glycosyltransferases (GTase) one by one by nonpolar mutations to assign functions to their gene products. The mutants expressed no OC or truncated OC oligosaccharides of different lengths. The truncated OC oligosaccharides revealed that the minimum structural requirements for the interactions of OC with bacteriophage phiR1-37, enterocoliticin, and OC-specific monoclonal antibody 2B5 were different. Furthermore, using chemical and structural analyses of the mutant LPSs, we could assign specific functions to all six GTases and also revealed the exact order in which the transferases build the hexasaccharide. Comparative modeling of the catalytic sites of glucosyltransferases WbcK and WbcL followed by site-directed mutagenesis allowed us to identify Asp-182 and Glu-181, respectively, as catalytic base residues of these two GTases. In general, conclusive evidence for specific GTase functions have been rare due to difficulties in accessibility of the appropriate donors and acceptors; however, in this work we were able to utilize the structural analysis of LPS to get direct experimental evidence for five different GTase specificities.

Defective B cell response to TLR9 ligand (CpG-ODN), Streptococcus pneumoniae and Haemophilus influenzae extracts in common variable immunodeficiency patients

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinaemia and antibody deficiency to both T dependent and independent antigens. Patients suffer from recurrent sinopulmonary infections mostly caused by Streptococcus pneumoniae and Haemophilus influenzae, but also gastrointestinal or autoimmune symptoms. Their response to vaccination is poor or absent. In this study we investigated B cell activation induced by the TLR9 specific ligand (CpG-ODN) and bacterial extracts from S. pneumoniae and H. influenzae known to stimulate several TLR. We found that B cells from CVID patients express lower levels of CD86 after stimulation with CpG-ODN, S. pneumoniae and H. influenzae extracts in combination with anti-IgM antibody and also display a lower proliferative index when stimulated with bacterial extracts. Our results point to a broad TLR signalling defect in B lymphocytes from CVID patients that may be related to the hypogammaglobulinaemia and poor response to vaccination characteristic of these patients.

Klebsiella pneumoniae triggers a cytotoxic effect on airway epithelial cells

Klebsiella pneumoniae is a capsulated Gram negative bacterial pathogen and a frequent cause of nosocomial infections. Despite its clinical relevance, little is known about the features of the interaction between K. pneumoniae and lung epithelial cells on a cellular level, neither about the role of capsule polysaccharide, one of its best characterised virulence factors, in this interaction.

Experimental pig yersiniosis to assess attenuation of Yersinia enterocolitica O:8 mutant strains

An experimental oral pig model was used to assess the pathogenic and immunogenic potential of Yersinia enterocolitica serotype O:8 wild-type strain 8081-L2 and its lipopolysaccharide (LPS) mutant derivatives: a spontaneous rough mutant 8081-R2, strain 8081-DeltawzzGB expressing O-antigen with uncontrolled chain lengths, and strain 8081-wbcEGB expressing semirough LPS with only one O-unit. Microbiological and immunological parameters of the infected pigs were followed from day 7 to 60 postinfection. The wild-type and all LPS mutant strains persisted in the lymphoid tissue of tonsils and small intestines, causing asymptomatic infection without any pathological changes. Although the pig is known as a reservoir of Yersiniae, a precise analysis of pathogenic and immunogenic parameters based on different in vitro tests (hematological response, killing ability of leukocytes and blood sera, antibody response, hydrogen peroxide production by macrophages, classical and alternative pathways of complement activation), revealed significant attenuation in the pathogenicity of the LPS mutant strains but not the loss of immunogenic potential. In comparison with the other strains, strain 8081-DeltawzzGB demonstrated more continuous leucocytosis with monocytosis, higher invasive potential, significant activation of hydrogen peroxide production by macrophages and an effective immunoglobulin G immune response accompanied by relevant histological immunomorphological rearrangements.

Comparison of Wavelet Transform and Time-Domain Analysis of Second Trimester Uterine Artery Doppler Waveforms in Screening for Pre-Eclampsia

The aim of this study was to compare time-domain waveform analysis of second-trimester uterine artery Doppler using the resistance index (RI) with waveform analysis using a mathematical tool known as wavelet transform for the prediction of pre-eclampsia (PE). This was a retrospective, nested case-cohort study of 336 women, 37 of whom subsequently developed PE. Uterine artery Doppler waveforms were analysed using both RI and waveform analysis. The utility of these indices in screening for PE was then evaluated using receiver operating characteristic curves. There were significant differences in uterine artery RI between the PE women and those with normal pregnancy outcome. After wavelet analysis, significant difference in the mean amplitude in wavelet frequency band 4 was noted between the 2 groups. The sensitivity for both Doppler RI and frequency band 4 for the detection of PE at a 10% false-positive rate was 45%. This small study demonstrates the application of wavelet transform analysis of uterine artery Doppler waveforms in screening for PE. Further prospective studies are needed in order to clearly define if this analytical approach to waveform analysis may have the potential to improve the detection of PE by uterine artery Doppler screening.

A theoretical examination of known and hypothetical clathrate hydrate materials

The recent synthesis of a new hydrogen binary hydrate with the sH structure has highlighted the potential storage capabilities of water clathrates [T. A. Strobel, C. A. Koh, and E. D. Sloan, J. Phys. Chem. B 112, 1885 (2008) and A. R. C. Duarte, A. Shariati, L. J. Rovetto, and C. J. Peters, J. Phys. Chem. B 112, 1888 (2008)]. In this work, the absorption of hydrogen and the promoters used in the experimental work are considered using a simplified model for the host-guest interaction, which allows one to understand the stabilizing effects of multiple help molecules. Two further hypothetical clathrates, which are isostructural with known zeolite structures, are also investigated. It is shown that the energy gained by absorbing adamantane into these two frameworks is far greater than that gained upon absorption of adamantane into the sH structure. Hence, a clathrate with the same topology as the DDR (Sigma 1) zeolite may be synthesizable with adamantane and hydrogen as guest molecules as, in the conditions explored here, this phase appears to be more stable than the sH structure. (C) 2009 American Institute of Physics. [DOI: 10.1063/1.3142503]

Large-scale spatial patterns in the distribution of Collembola (Hexapoda) species in Antarctic terrestrial ecosystems

We tested whether the distribution of three common springtail species (Gressittacantha terranova, Gomphiocephalus hodgsoni and Friesea grisea) in Victoria Land (Antarctica) could be modelled as a function of latitude, longitude, altitude and distance from the sea.

Victoria Land, Ross Dependency, Antarctica.

Generalized linear models were constructed using species presence/absence data relative to geographical features (latitude, longitude, altitude, distance from sea) across the species' entire ranges. Model results were then integrated with the known phylogeography of each species and hypotheses were generated on the role of climate as a major driver of Antarctic springtail distribution.

Based on model selection using Akaike's information criterion, the species' distributions were: hump-shaped relative to longitude and monotonic with altitude for Gressittacantha terranova; hump-shaped relative to latitude and monotonic with altitude for Gomphiocephalus hodgsoni; and hump-shaped relative to longitude and monotonic with latitude, altitude and distance from the sea for Friesea grisea.

No single distributional pattern was shared by the three species. While distributions were partially a response to climatic spatial clines, the patterns observed strongly suggest that past geological events have influenced the observed distributions. Accordingly, present-day spatial patterns are likely to have arisen from the interaction of historical and environmental drivers. Future studies will need to integrate a range of spatial and temporal scales to further quantify their respective roles.

Known risk factors for violence predict 12-month-old infants' aggressiveness with peers

This study tested the hypothesis that 12-month-old infants' use of force against peers is associated with known risk factors for violence. We conducted a prospective longitudinal study, which included laboratory observations of firstborn British infants (N = 271) during simulated birthday parties. No gender differences in aggressiveness were observed. The infants' observed aggressiveness was significantly correlated with mothers' mood disorder during pregnancy and with mothers' history of conduct problems. Infants' observed aggressiveness was correlated with parents' ratings of infants' anger and aggression, which were also predicted by mothers' mood disorder and history of conduct problems. Our findings indicate that infants at risk for serious aggression can already be identified when the motor ability to use physical force first enters the human repertoire.

Cannibale, Frigidaire, and the Multitude: Post-1977 Italian Comics through Radical Theory

A direct connection between comics and contemporary critical theory is to be found in the activity of the collective of artists who created "Cannibale" and "Frigidaire", the two most innovative Italian comics magazines of the late 1970s and early 1980s. The work of Andrea Pazienza, Filippo Scòzzari, Stefano Tamburini, Tanino Liberatore and Massimo Mattioli should be regarded as an expression of the radical movements from which Marxist Autonomist thinkers such as Antonio Negri, Franco ‘Bifo’ Berardi and Paolo Virno also emerged in the same period. As a consequence, the writings of the latter can be used to analyse the narrative and visual style, the recurring themes, and the editorial characteristics of the comics of the former. Moreover, an interesting parallel can also be drawn between the work of the most influential of these artists, Andrea Pazienza, and the thought of another prominent Italian philosopher, Giorgio Agamben, whose earliest books were published at that time too. The aim of this article is to show that the ideas of a number of the thinkers who have recently become internationally known as representatives of radical Italian theory, are useful to understand the work of this group of comics authors.

Arsenic Bioaccessibility in Cooked Rice as Affected by Arsenic in Cooking Water

Rice can easily accumulate arsenic (As) into its grain and is known to be the highest As-containing cereal. In addition, the As burden in rice may increase during its processing (such as when cooking using As-polluted water). The health risk posed by the presence of As in cooked rice depends on its release from the matrix along the digestive system (bioaccessibility). Two types of white polished long-grain rice, namely, nonparboiled and parboiled (total As: 202 and 190 mu g As kg(-1), respectively), were cooked in excess of water with different levels of As (0, 10, 47, 222, and 450 mu g As L-1). The bioaccessibility of As from these cooked rice batches was evaluated with an in vitro dynamic digestion process. Rice cooked with water containing 0 and 10 mu g As L-1 showed lower As concentrations than the raw (uncooked) rice. However, cooking water with relatively high As content (>= 47 mu g As L-1) significantly increased the As concentration in the cooked rice up to 8- and 9-fold for the nonparboiled and parboiled rice, respectively. Parboiled rice, which is most widely consumed in South Asia, showed a higher percentage of As bioaccessibility (59% to 99%) than nonparboiled rice (36% to 69%) and most of the As bioaccessible in the cooked rice (80% to 99%) was released easily during the first 2 h of digestion. The estimation of the As intake through cooked rice based on the As bioaccessibility highlights that a few grams of cooked rice (less than 25 g dry weight per day) cooked with highly As contaminated water is equivalent to the amount of As from 2 L water containing the maximum permissible limit (10 mu g As L-1).

Whole-mitochondrial genome sequencing in primary open-angle glaucoma using massively parallel sequencing identifies novel and known pathogenic variants

Purpose:The aim of this study was to determine whether mutations in mitochondrial DNA play a role in high-pressure primary open-angle glaucoma (OMIM 137760) by analyzing new data from massively parallel sequencing of mitochondrial DNA.
Methods:Glaucoma patients with high-tension primary open-angle glaucoma and ethnically matched and age-matched control subjects without glaucoma were recruited. The entire human mitochondrial genome was amplified in two overlapping fragments by long-range polymerase chain reaction and used as a template for massively parallel sequencing on an Ion Torrent Personal Genome Machine. All variants were confirmed by conventional Sanger sequencing.
Results:Whole-mitochondrial genome sequencing was performed in 32 patients with primary open-angle glaucoma from India (n = 16) and Ireland (n = 16). In 16 of the 32 patients with primary open-angle glaucoma (50% of cases), there were 22 mitochondrial DNA mutations consisting of 7 novel mutations and 8 previously reported disease-associated sequence variants. Eight of 22 (36.4%) of the mitochondrial DNA mutations were in complex I mitochondrial genes.
Conclusion:Massively parallel sequencing using the Ion Torrent Personal Genome Machine with confirmation by Sanger sequencing detected a pathogenic mitochondrial DNA mutation in 50% of the primary open-angle glaucoma cohort. Our findings support the emerging concept that mitochondrial dysfunction results in the development of glaucoma and, more specifically, that complex I defects play a significant role in primary open-angle glaucoma pathogenesis.