Investigating The Sequence Diversity Of Transferrin Binding Protein B In Haemophilus Influenzae

BACKGROUND Haemophilus influenzae (Hi) is a Gram-negative bacterium that is exclusive to the upper respiratory tract of humans. Hi strains possessing an extracellular polysaccharide capsule, particularly serotype b H. influenzae (Hib), are responsible for invasive infections such as bacterial meningitis and bacteremia. Strains without a polysaccharide capsule, known as non-typable H. influenzae (NTHi), are responsible for ear infections and other diseases in children. Disease caused by Hib infections has become rare in developed countries since the introduction of a conjugate serogroup b vaccine. However disease caused by NTHi and other Hi serotypes, particularly type a Hi, has increased [1]. To prevent disease caused by non-vaccine Hi serotypes novel vaccines will need to be produced that are effective against multiple serotypes of Hi, including NTHi. RATIONALE It has been demonstrated that transferrin binding protein B (TbpB) in the porcine pathogen, Actinobacillus pleuropneumoniae (Ap), is essential for survival and causing disease in a porcine infection model [2]. TbpB is now being developed as a vaccine target. Since Ap and Hi are both host-restricted bacteria in the Pasteurellaceae family, we consider TbpB in Hi as a logical vaccine target. METHODS To investigate the diversity of TbpBs in Hi, the tbpB genes from a collection of 43 unique Hi strains from Canada, the United States and England were sequenced. These strains represent a wide diversity of clinical manifestations and serotypes, allowing us to investigate the diversity of the tbpB gene in Hi. Phylogenetic analysis was undertaken to cluster the tbpB genes based on sequence similarity. These analyses of the relationships between phylogenetic clusters and clinical/epidemiological information explore the prevalence and diversity of transferrin receptor genes in Hi. RESULTS Analysis of the data showed that the sequences of the various TbpB proteins clustered independently of serotype. This shows that development of a TbpB-based vaccine would logically target all serotypes and non-typeable strains, rather than having to develop a series of vaccines for the different groups. The analysis of the multiple sequence alignments generated from the TbpB sequences showed higher conservation in the C lobe of TbpB than the N lobe.  Of particular interest was the observation made while mapping conserved regions of the sequence alignments to a structural model of TbpB that a region exists on the N lobe of TbpB which is highly conserved in all Hi strains sequenced. FUTURE DIRECTIONS The immunogenic properties of this conserved region of TbpB have not yet been investigated. The number of strains sequenced was also limited and they were only of serotype B and NTHi. More diverse Hi strains need to be sequenced to confirm the conservation of TbpB across all serotypes and further studies still need to be completed to confirm that these conserved regions are actually immunogenic. However this study has shown that TbpB has potential to be used as a target for a new cross protective Hi vaccine

Experiments In Layered Electro-Photographic Printing 267

Electro-photographic printing processes employed by products such as laser printers and photocopiers are commonly used to deposit and fuse thin layers of thermoplastic powder onto paper. This report describes preliminary experiments aimed at adapting the electro-photographic printing process for use as a layered manufacturing technique. 3-D electro-photographic printing holds considerable potential as an inexpensive freeform fabrication technique that is suitable for office environments. The possibilities for selective coloring are also discussed.Mechanical Engineerin

A Single Tri-Epitopic Antibody Virtually Recapitulates the Potency of a Combination of Three Monoclonal Antibodies in Neutralization of Botulinum Neurotoxin Serotype A.

The standard of treatment for botulism, equine antitoxin, is a foreign protein with associated safety issues and a short serum half-life which excludes its use as a prophylactic antitoxin and makes it a less-than-optimal therapeutic. Due to these limitations, a recombinant monoclonal antibody (mAb) product is preferable. It has been shown that combining three mAbs that bind non-overlapping epitopes leads to highly potent botulinum neurotoxin (BoNT) neutralization. Recently, a triple human antibody combination for BoNT/A has demonstrated potent toxin neutralization in mouse models with no serious adverse events when tested in a Phase I clinical trial. However, a triple antibody therapeutic poses unique development and manufacturing challenges. Thus, potentially to streamline development of BoNT antitoxins, we sought to achieve the potency of multiple mAb combinations in a single IgG-based molecule that has a long serum half-life. The design, production, and testing of a single tri-epitopic IgG1-based mAb (TeAb) containing the binding sites of each of the three parental BoNT/A mAbs yielded an antibody of nearly equal potency to the combination. The approach taken here could be applied to the design and creation of other multivalent antibodies that could be used for a variety of applications, including toxin elimination

The Dictyostelium discoideum genome lacks significant DNA methylation and uncovers palindromic sequences as a source of false positives in bisulfite sequencing

DNA methylation, the addition of a methyl (CH3) group to a cytosine residue, is an evolutionarily conserved epigenetic mark involved in a number of different biological functions in eukaryotes, including transcriptional regulation, chromatin structural organization, cellular differentiation and development. In the social amoeba Dictyostelium, previous studies have shown the existence of a DNA methyltransferase (DNMA) belonging to the DNMT2 family, but the extent and function of 5-methylcytosine in the genome are unclear. Here, we present the whole genome DNA methylation profile of Dictyostelium discoideum using deep coverage replicate sequencing of bisulfite-converted gDNA extracted from post-starvation cells. We find an overall very low number of sites with any detectable level of DNA methylation, occurring at significant levels in only 303-3432 cytosines out of the ∼7.5 million total cytosines in the genome depending on the replicate. Furthermore, a knockout of the DNMA enzyme leads to no overall decrease in DNA methylation. Of the identified sites, significant methylation is only detected at 11 sites in all four of the methylomes analyzed. Targeted bisulfite PCR sequencing and computational analysis demonstrate that the methylation profile does not change during development and that these 11 cytosines are most likely false positives generated by protection from bisulfite conversion due to their location in hairpin-forming palindromic DNA sequences. Our data therefore provide evidence that there is no significant DNA methylation in Dictyostelium before fruiting body formation and identify a reproducible experimental artifact from bisulfite sequencing. © 2023 The Author(s). Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics

Thermal and chemical unfolding and refolding of a eukaryotic sodium channel

Voltage-gated sodium channels are dynamic membrane proteins essential for signaling in nervous and muscular systems. They undergo substantial conformational changes associated with the closed, open and inactivated states. However, little information is available regarding their conformational stability. In this study circular dichroism spectroscopy was used to investigate the changes in secondary structure accompanying chemical and thermal denaturation of detergent-solubilised sodium channels isolated from Electrophorus electricus electroplax. The proteins appear to be remarkably resistant to either type of treatment, with "denatured" channels, retaining significant helical secondary structure even at 77 degrees C or in 10% SDS. Further retention of helical secondary structure at high temperature was observed in the presence of the channel-blocking tetrodotoxin. It was possible to refold the thermally-denatured (but not chemically-denatured) channels in vitro. The correctly refolded channels were capable of undergoing the toxin-induced conformational change indicative of ligand binding. In addition, flux measurements in liposomes showed that the thermally-denatured (but not chemically-denatured) proteins were able to re-adopt native, active conformations. These studies suggest that whilst sodium channels must be sufficiently flexible to undergo major conformational changes during their functional cycle, the proteins are highly resistant to unfolding, a feature that is important for maintaining structural integrity during dynamic processes. (c) 2009 Elsevier B.V. All rights reserved

microRNAs and the evolution of complex multicellularity:Identification of a large, diverse complement of microRNAs in the brown alga Ectocarpus

There is currently convincing evidence that microRNAs have evolved independently in at least six different eukaryotic lineages: animals, land plants, chlorophyte green algae, demosponges, slime molds and brown algae. MicroRNAs from different lineages are not homologous but some structural features are strongly conserved across the eukaryotic tree allowing the application of stringent criteria to identify novel microRNA loci. A large set of 63 microRNA families was identified in the brown alga Ectocarpus based on mapping of RNA-seq data and nine microRNAs were confirmed by northern blotting. The Ectocarpus microRNAs are highly diverse at the sequence level with few multi-gene families, and do not tend to occur in clusters but exhibit some highly conserved structural features such as the presence of a uracil at the first residue. No homologues of Ectocarpus microRNAs were found in other stramenopile genomes indicating that they emerged late in stramenopile evolution and are perhaps specific to the brown algae. The large number of microRNA loci in Ectocarpus is consistent with the developmental complexity of many brown algal species and supports a proposed link between the emergence and expansion of microRNA regulatory systems and the evolution of complex multicellularity

The genomic repertoire for cell cycle control and DNA metabolism in S. purpuratus

A search of the Strongylocentrotus purpuratus genome for genes associated with cell cycle control and DNA metabolism shows that the known repertoire of these genes is conserved in the sea urchin, although with fewer family members represented than in vertebrates, and with some cases of echinoderm-specific gene diversifications. For example, while homologues of the known cyclins are mostly encoded by single genes in S. purpuratus (unlike vertebrates, which have multiple isoforms), there are additional genes encoding novel cyclins of the B and K/L types. Almost all known cyclin-dependent kinases (CDKs) or CDK-like proteins have an orthologue in S. purpuratus; CDK3 is one exception, whereas CDK4 and 6 are represented by a single homologue, referred to as CDK4. While the complexity of the two families of mitotic kinases, Polo and Aurora, is close to that found in the nematode, the diversity of the NIMA-related kinases (NEK proteins) approaches that of vertebrates. Among the nine NEK proteins found in S. purpuratus, eight could be assigned orthologues in vertebrates, whereas the ninth is unique to sea urchins. Most known DNA replication, DNA repair and mitotic checkpoint genes are also present, as are homologues of the pRB (two) and p53 (one) tumor suppressors. Interestingly, the p21/p27 family of CDK inhibitors is represented by one homologue, whereas the INK4 and ARF families of tumor suppressors appear to be absent, suggesting that these evolved only in vertebrates. Our results suggest that, while the cell cycle control mechanisms known from other animals are generally conserved in sea urchin, parts of the machinery have diversified within the echinoderm lineage. The set of genes uncovered in this analysis of the S. purpuratus genome should enhance future research on cell cycle control and developmental regulation in this model

Perfectionism and Grit in Competitive Sport

Perfectionism and grit have both been linked to the achievement-striving process in sport, yet very little is known about the relationships between the two constructs. The present study explored the degree to which perfectionistic strivings and perfectionistic concerns predicted two dimensions of grit—consistency of interest and perseverance of effort—in a sample of 251 intercollegiate varsity athletes (Mage = 20.34 years; SD = 2.0). Both perfectionism and grit were conceptualized and measured as multidimensional domain-specific constructs. Results of structural equation modeling analyses indicated that perfectionistic strivings was positively associated with consistency of interest (β = .49, p < .001) and perseverance of effort (β = .92, p < .001). In contrast, perfectionistic concerns was negatively associated with both consistency of interest (β = -.47, p < .001) and perseverance of effort (β = -.66, p < .001). Results indicate that higher-order dimensions of perfectionism (i.e., perfectionistic strivings and perfectionistic concerns) are associated with domain-specific aspects of grit in sport. Results highlight the importance of (a) differentiating between athletes’ perfectionistic strivings and perfectionistic concerns in sport, and (b) treating consistency of interest and perseverance of effort as separate components of grit. Future research that examines the combined effects of perfectionism and grit on the achievement-striving process in competitive sport is recommended