Fasciola hepatica cathepsin L-like proteases: biology, function and potential in the development of first generation liver fluke vaccines.

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines. Author Keywords: Helminths; Trematodes; Parasites; Cathepsins; Proteases; Vaccines; Immunology; Biochemistry

The antimicrobial effects of root canal irrigation and medication

The role of microorganisms in the development and maintenance of pulpal and periapical inflammation have been well documented. The success of root canal treatment largely depends on the elimination of microbial contamination from the root canal system. Although mechanical instrumentation of root canals can reduce bacterial population, effective elimination of bacteria cannot be achieved without the use of antimicrobial root canal irrigation and medication. This review will discuss the antimicrobial effects of the known root canal irrigants and medicaments and explore future developments in the field. © 2007 Mosby, Inc. All rights reserved.

Antimicrobial activity of truncated alpha-defensin (human neutrophil peptide (HNP)-1) analogues without disulphide bridges.

Antimicrobial peptides play an important role in host defence, particularly in the oral cavity where there is constant challenge by microorganisms. The a-defensin antimicrobial peptides comprise 30–50% of the total protein in the azurophilic granules of human neutrophils, the most abundant of which is human neutrophil peptide 1 (HNP-1). Despite its antimicrobial activity, a limiting factor in the potential therapeutic use of HNP-1 is its chemical synthesis with the correct disulphide topology. In the present study, we synthesised a range of truncated defensin analogues lacking disulphide bridges. All the analogues were modelled on the C-terminal region of HNP-1 and their antimicrobial activity was tested against a range of microorganisms, including oral pathogens. Although there was variability in the antimicrobial activity of the truncated analogues synthesised, a truncated peptide named 2Abz23S29 displayed a broad spectrum of antibacterial activity, effectively killing all the bacterial strains tested. The finding that truncated peptides, modelled on the C-terminal ß-hairpin region of HNP-1 but lacking disulphide bridges, display antimicrobial activity could aid their potential use in therapeutic interventions.

Geophysical imaging of stimulated microbial biomineralization

Understanding how microorganisms influence the physical and chemical properties of the subsurface is hindered by our inability to observe microbial dynamics in real time and with high spatial resolution. Here, we investigate the use of noninvasive geophysical methods to monitor biomineralization at the laboratory scale during stimulated sulfate reduction under dynamic flow conditions. Alterations in sediment characteristics resulting from microbe-mediated sulfide mineral precipitation were concomitant with changes in complex resistivity and acoustic wave propagation signatures. The sequestration of zinc and iron in insoluble sulfides led to alterations in the ability of the pore fluid to conduct electrical charge and of the saturated sediments to dissipate acoustic energy. These changes resulted directly from the nucleation, growth, and development of nanoparticulate precipitates along grain surfaces and within the pore space. Scanning and transmission electron microscopy (SEM and TEM) confirmed the sulfides to be associated with cell surfaces, with precipitates ranging from aggregates of individual 3-5 nm nanocrystals to larger assemblages of up to 10-20 m in diameter. Anomalies in the geophysical data reflected the distribution of mineral precipitates and biomass over space and time, with temporal variations in the signals corresponding to changes in the aggregation state of the nanocrystalline sulfides. These results suggest the potential for using geophysical techniques to image certain subsurface biogeochemical processes, such as those accompanying the bioremediation of metal-contaminated aquifers.

Performance of a Sequential Reactive Barrier for Bioremediation of Coal Tar Contaminated Groundwater

Following a thorough site investigation, a biological Sequential Reactive Barrier (SEREBAR), designed to remove Polycyclic Aromatic Hydrocarbons (PAHs) and BTEX compounds, was installed at a Former Manufactured Gas Plant (FMGP) site. The novel design of the barrier comprises, in series, an interceptor and six reactive chambers. The first four chambers (2 nonaerated-2 aerated) were filled with sand to encourage microbial colonization. Sorbant Granular Activated Carbon (GAC) was present in the final two chambers in order to remove any recalcitrant compounds. The SEREBAR has been in continuous operation for 2 years at different operational flow rates (ranging from 320 L/d to 4000 L/d, with corresponding residence times in each chamber of 19 days and 1.5 days, respectively). Under low flow rate conditions (320-520 L/d) the majority of contaminant removal (>93%) occurred biotically within the interceptor and the aerated chambers. Under high flow rates (1000-4000 L/d) and following the installation of a new interceptor to prevent passive aeration, the majority of contaminant removal (>80%) again occurred biotically within the aerated chambers. The sorption zone (GAC) proved to be an effective polishing step, removing any remaining contaminants to acceptable concentrations before discharge down-gradient of the SEREBAR (overall removals >95%).

Compact quadrifilar helix antenna

Introduction: The quadrifilar helix antenna (QHA) is used widely for terrestrial [1] and space communication systems [2], where it is necessary to generate a circularly polarised cardioid-shaped radiation pattern with a high front-to-back ratio and low cross-polarisation. The radiating structure comprises four helical conductors which are excited in phase quadrature at the feed point, which is usually located at the centre of the top radials. The physical size of the quadrifilar antenna can be reduced by dielectric loading [3] or by meandering the printed linear elements [4]. However, in the former arrangement dielectric absorption reduces the radiation efficiency of the antenna, and the latter technique is not suitable for constructing free standing wire structures, which are normally used for spacecraft payloads in the VHF and UHF bands [2]. This Letter shows that a significant reduction in the axial length of a 1/2 turn half-wavelength QHA can be achieved by modifying the geometry of the helices in the region around the midpoint where a current null exists. Simulated and experimental results at L band are used to show that a size reduction of up to 15% is possible without significantly degrading the pattern shape and the bandwidth.

Selection with melphalan or paclitaxel (Taxol) yields variants with different patterns of multidrug resistance, integrin expression and in vitro invasiveness

A melphalan-resistant variant (Roswell Park Memorial Institute (RPMI)-2650M1) and a paclitaxel-resistant variant (RPMI-1650Tx) of the drug-sensitive human nasal carcinoma cell line, RPMI-2650. were established. The multidrug resistance (MDR) phenotype in the RPMI-2650Tx appeared to be P-glycoprotein (PgP)-mediated. Overexpression of multidrug resistant protein (MRP) family members was observed in the RPMI-2650M1 cells, which were also much more invasive in vitro than the parental cell line or the paclitaxel-resistant variant. Increased expression of alpha (2), alpha (5), alpha (6), beta (1) and beta (4) integrin subunits, decreased expression of alpha (4) integrin subunit, stronger adhesion to collagen type IV, laminin, fibronectin and matrigel, increased expression of MMP-2 and MMP-9 and significant motility compared with the parental cells were observed, along with a high invasiveness in the RPMI-7650M1 cells. Decreased expression of the alpha (2) integrin subunit, decreased attachment to collagen type IV, absence of cytokeratin 18 expression, no detectable expression of gelatin-degrading proteases and poor motility may be associated with the non-invasiveness of the RPMI-2650Tx variant. These results suggest that melphalan exposure can result in not only a MDR phenotype. but could also make cancer cells more invasive, whereas paclitaxel exposure resulted in MDR without increasing the in vitro invasiveness in the RPMI-2650 cells. (C) 2001 Elsevier Science Ltd. All rights reserved.

A nonradioactive method for the assay of polyphosphate kinase activity and its application in the study of polyphosphate metabolism in Burkholderia cepacia

Studies of polyphosphate (polyP) metabolism in microorganisms have been hampered by the lack of a convenient method for the assay in cell extracts of the activity of polyphosphate kinase (PPK), the enzyme principally responsible for microbial polyP biosynthesis. We report the development of such an assay, based on the well-established metachromatic reaction, with toluidine blue, of the polyP formed during the PPK-catalyzed reaction. The method was successfully used in the characterization of PPK activity in crude extracts of an environmental Burkholderia cepacia isolate. The development of a protocol for the physical recovery of polyP from solution is also reported. (C) 2002 Elsevier Science (USA). All rights reserved.

Isolation and characterisation of bacterial strains containing enantioselective DMSO reductase activity: application to the kinetic resolution of racemic sulfoxides

The kinetic resolution of racemic sulfoxides by dimethyl sulfoxide (DMSO) reductases was investigated with a range of microorganisms. Three bacterial isolates (provisionally identified as Citrobacter braakii, Klebsiella sp. and Serratia sp.) expressing DMSO reductase activity were isolated from environmental samples by anaerobic enrichment with DMSO as terminal electron acceptor. The organisms reduced a diverse range of racemic sulfoxides to yield either residual enantiomer depending upon the strain used. C. braakii DMSO-11 exhibited wide substrate specificity that included dialkyl, diaryl and alkylaryl sulfoxides, and was unique in its ability to reduce the thiosulfinate 1,4-dihydrobenzo-2, 3-dithian-2-oxide. DMSO reductase was purified from the periplasmic fraction of C. braakii DMSO-11 and was used to demonstrate unequivocally that the DMSO reductase was responsible for enantiospecific reductive resolution of racemic sulfoxides.

Genome sequence of the metazoan plant-parasitic nematode Meloidogyne incognita

Plant-parasitic nematodes are major agricultural pests worldwide and novel approaches to control them are sorely needed. We report the draft genome sequence of the root-knot nematode Meloidogyne incognita, a biotrophic parasite of many crops, including tomato, cotton and coffee. Most of the assembled sequence of this asexually reproducing nematode, totaling 86 Mb, exists in pairs of homologous but divergent segments. This suggests that ancient allelic regions in M. incognita are evolving toward effective haploidy, permitting new mechanisms of adaptation. The number and diversity of plant cell wall-degrading enzymes in M. incognita is unprecedented in any animal for which a genome sequence is available, and may derive from multiple horizontal gene transfers from bacterial sources. Our results provide insights into the adaptations required by metazoans to successfully parasitize immunocompetent plants, and open the way for discovering new antiparasitic strategies.