Characterization of putative sialidase genes in Gardnerella vaginalis

Gardnerella vaginalis is a hallmark organism in the dysbiosis bacterial vaginosis (BV) in reproductive age women although its role in this condition is not currently understood. Diversity within G. vaginalis in terms of virulence factors such as sialidase activity, may explain why it is also observed in asymptomatic women. This thesis aimed to identify genomic determinants of sialidase activity in G. vaginalis and better understand its role in BV. G. vaginalis has demonstrated genotypic and phenotypic diversity in research over the years and has been divided into four subgroups (A-D) based on cpn60 universal target sequencing. Recent research has demonstrated that a previously identified sialidase gene (Gene 1) does not correlate with sialidase activity. Analysis of 39 available G. vaginalis genome sequences identified a second sialidase gene (Gene 2), and its presence correlated with sialidase activity in 112 G. vaginalis isolates. Based on examination of the predicted amino acid sequences of the two sialidases, we hypothesized that Gene 1 encodes an intracellular sialidase while Gene 2 encodes an extracellular sialidase found almost exclusively in subgroup B strains. Gene 1 was shown to encode for a sialidase enzyme with a pH optimum of 4.5 to 5.0. No protein could be expressed from Gene 2 in E. coli. A homopolymer region was identified in Gene 2 that caused an early stop codon, and may be involved in slipped-strand mispairing. When sialidase activity was assayed in cultures of sialidase activity positive (Gene 2 positive) and negative (Gene 2 negative) isolates, activity was detected only in the cell pellet of Gene 2 positive isolates, suggesting that Protein 2 is a cell bound, extracellular sialidase. The results of this thesis demonstrate that G. vaginalis has at least two sialidase genes, only one of which encodes the extracellular sialidase activity observed in some isolates. This may help explain why G. vaginalis is found in women with symptomatic BV, as well as women with no signs of dysbiosis. The finding that extracellular sialidase activity is confined to subgroup B G. vaginalis suggests that these isolates may have an important role in the establishment and maintenance of vaginal dysbiosis

Gardnerella vaginalis subgroups defined by cpn60 sequencing and sialidase activity in isolates from Canada, Belgium and Kenya

Increased abundance of Gardnerella vaginalis and sialidase activity in vaginal fluid is associated with bacterial vaginosis (BV), a common but poorly understood clinical entity associated with poor reproductive health outcomes. Since most women are colonized with G. vaginalis, its status as a normal member of the vaginal microbiota or pathogen causing BV remains controversial, and numerous classification schemes have been described. Since 2005, sequencing of the chaperonin-60 universal target (cpn60 UT) has distinguished four subgroups in isolate collections, clone libraries and deep sequencing datasets. To clarify potential clinical and diagnostic significance of cpn60 subgroups, we undertook phenotypic and molecular characterization of 112 G. vaginalis isolates from three continents. A total of 36 subgroup A, 33 B, 35 C and 8 D isolates were identified through phylogenetic analysis of cpn60 sequences as corresponding to four "clades" identified in a recently published study, based on sequencing 473 genes across 17 isolates. cpn60 subgroups were compared with other previously described molecular methods for classification of Gardnerella subgroups, including amplified ribosomal DNA restriction analysis (ARDRA) and real-time PCR assays designed to quantify subgroups in vaginal samples. Although two ARDRA patterns were observed in isolates, each was observed in three cpn60 subgroups (A/B/D and B/C/D). Real-time PCR assays corroborated cpn60 subgroups overall, but 13 isolates from subgroups A, B and D were negative in all assays. A putative sialidase gene was detected in all subgroup B, C and D isolates, but only in a single subgroup A isolate. In contrast, sialidase activity was observed in all subgroup B isolates, 3 (9%) subgroup C isolates and no subgroup A or D isolates. These observations suggest distinct roles for G. vaginalis subgroups in BV pathogenesis. We conclude that cpn60 UT sequencing is a robust approach for defining G. vaginalis subgroups within the vaginal microbiome

High-speed focal modulation microscopy using acousto-optical modulators

Focal Modulation Microscopy (FMM) is a single-photon excitation fluorescence microscopy technique which effectively rejects the out-of-focus fluorescence background that arises when imaging deep inside biological tissues. Here, we report on the implementation of FMM in which laser intensity modulation at the focal plane is achieved using acousto-optic modulators (AOM). The modulation speed is greatly enhanced to the MHz range and thus enables real-time image acquisition. The capability of FMM is demonstrated by imaging fluorescence labeled vasculatures in mouse brain as well as self-made tissue phantom

Filoviruses utilize glycosaminoglycans for their attachment to target cells

Filoviruses are the cause of severe hemorrhagic fever in human and nonhuman primates. The envelope glycoprotein (GP), responsible for both receptor binding and fusion of the virus envelope with the host cell membrane, has been demonstrated to interact with multiple molecules in order to enhance entry into host cells. Here we have demonstrated that filoviruses utilize glycosaminoglycans, and more specifically heparan sulfate proteoglycans, for their attachment to host cells. This interaction is mediated by GP and does not require the presence of the mucin domain. Both the degree of sulfation and the structure of the carbohydrate backbone play a role in the interaction with filovirus GPs. This new step of filovirus interaction with host cells can potentially be a new target for antiviral strategies. As such, we were able to inhibit filovirus GP-mediated infection using carrageenan, a broad-spectrum microbicide that mimics heparin, and also using the antiviral dendrimeric peptide SB105-A10, which interacts with heparan sulfate, antagonizing the binding of the virus to cells

The origin of the light distribution in spiral galaxies

We analyse a high-resolution, fully cosmological, hydrodynamical disc galaxy simulation, to study the source of the double-exponential light profiles seen in many stellar discs, and the effects of stellar radial migration upon the spatiotemporal evolution of both the disc age and metallicity distributions. We find a ‘break’ in the pure exponential stellar surface brightness profile, and trace its origin to a sharp decrease in the star formation per unit surface area, itself produced by a decrease in the gas volume density due to a warping of the gas disc. Star formation in the disc continues well beyond the break. We find that the break is more pronounced in bluer wavebands. By contrast, we find little or no break in the mass density profile. This is, in part, due to the net radial migration of stars towards the external parts of the disc. Beyond the break radius, we find that ∼60 per cent of the resident stars migrated from the inner disc, while ∼25 per cent formed in situ. Our simulated galaxy also has a minimum in the age profile at the break radius but, in disagreement with some previous studies, migration is not the main mechanism producing this shape. In our simulation, the disc metallicity gradient flattens with time, consistent with an ‘inside-out’ formation scenario. We do not find any difference in the intensity or the position of the break with inclination, suggesting that perhaps the differences found in empirical studies are driven by dust extinction

Temporal trends in serum concentrations of polychlorinated dioxins, furans, and PCBs among adult women living in Chapaevsk, Russia: a longitudinal study from 2000 to 2009

<p>Abstract</p> <p>Background</p> <p>The present study assessed the temporal trend in serum concentrations of polychlorinated dibenzo-<it>p</it>-dioxins, dibenzofurans, and biphenyls (PCBs) among residents of a Russian town where levels of these chemicals are elevated due to prior industrial activity.</p> <p>Methods</p> <p>Two serum samples were collected from eight adult women (in 2000 and 2009), and analyzed with gas chromatography-high-resolution mass spectrometry.</p> <p>Results</p> <p>The average total toxic equivalency (TEQ) decreased by 30% (from 36 to 25 pg/g lipid), and the average sum of PCB congeners decreased by 19% (from 291 to 211 ng/g lipid). Total TEQs decreased for seven of the eight women, and the sum of PCBs decreased for six of eight women. During this nine year period, larger decreases in serum TEQs and PCBs were found in women with greater increases in body mass index.</p> <p>Conclusions</p> <p>This study provides suggestive evidence that average serum concentrations of dioxins, furans, and PCBs are decreasing over time among residents of this town.</p

Simultaneous Effects of Light Intensity and Phosphorus Supply on the Sterol Content of Phytoplankton

Sterol profiles of microalgae and their change with environmental conditions are of great interest in ecological food web research and taxonomic studies alike. Here, we investigated effects of light intensity and phosphorus supply on the sterol content of phytoplankton and assessed potential interactive effects of these important environmental factors on the sterol composition of algae. We identified sterol contents of four common phytoplankton genera, Scenedesmus, Chlamydomonas, Cryptomonas and Cyclotella, and analysed the change in sterol content with varying light intensities in both a high-phosphorus and a low-phosphorus approach. Sterol contents increased significantly with increasing light in three out of four species. Phosphorus-limitation reversed the change of sterol content with light intensity, i.e., sterol content decreased with increasing light at low phosphorus supply. Generally sterol contents were lower in low-phosphorus cultures. In conclusion, both light and phosphorus conditions strongly affect the sterol composition of algae and hence should be considered in ecological and taxonomic studies investigating the biochemical composition of algae. Data suggest a possible sterol limitation of growth and reproduction of herbivorous crustacean zooplankton during summer when high light intensities and low phosphorus supply decrease sterol contents of algae

Investigation of the Origin and Spread of a Mammalian Transposable Element Based on Current Sequence Diversity

Almost half the human genome consists of mobile DNA elements, and their analysis is a vital part of understanding the human genome as a whole. Many of these elements are ancient and have persisted in the genome for tens or hundreds of millions of years, providing a window into the evolution of modern mammals. The Golem family have been used as model transposons to highlight computational analyses which can be used to investigate these elements, particularly the use of molecular dating with large transposon families. Whole-genome searches found Golem sequences in 20 mammalian species. Golem A and B subsequences were only found in primates and squirrel. Interestingly, the full-length Golem, found as a few copies in many mammalian genomes, was found abundantly in horse. A phylogenetic profile suggested that Golem originated after the eutherian–metatherian divergence and that the A and B subfamilies originated at a much later date. Molecular dating based on sequence diversity suggests an early age, of 175 Mya, for the origin of the family and that the A and B lineages originated much earlier than expected from their current taxonomic distribution and have subsequently been lost in some lineages. Using publically available data, it is possible to investigate the evolutionary history of transposon families. Determining in which organisms a transposon can be found is often used to date the origin and expansion of the families. However, in this analysis, molecular dating, commonly used for determining the age of gene sequences, has been used, reducing the likelihood of errors from deleted lineages

The relationship between cadence, pedalling technique and gross efficiency in cycling

Technique and energy saving are two variables often considered as important for performance in cycling and related to each other. Theoretically, excellent pedalling technique should give high gross efficiency (GE). The purpose of the present study was to examine the relationship between pedalling technique and GE. 10 well-trained cyclists were measured for GE, force effectiveness (FE) and dead centre size (DC) at a work rate corresponding to ~75% of VO2max during level and inclined cycling, seat adjusted forward and backward, at three different cadences around their own freely chosen cadence (FCC) on an ergometer. Within subjects, FE, DC and GE decreased as cadence increased (p < 0.001). A strong relationship between FE and GE was found, which was to great extent explained by FCC. The relationship between cadence and both FE and GE, within and between subjects, was very similar, irrespective of FCC. There was no difference between level and inclined cycling position. The seat adjustments did not affect FE, DC and GE or the relationship between them. Energy expenditure is strongly coupled to cadence, but force effectiveness, as a measure for pedalling technique, is not likely the cause of this relationship. FE, DC and GE are not affected by body orientation or seat adjustments, indicating that these parameters and the relationship between them are robust to coordinative challenges within a range of cadence, body orientation and seat position that is used in regular cycling