Deformability Assessment of Waterborne Protozoa Using a Microfluidic-Enabled Force Microscopy Probe

Many modern filtration technologies are incapable of the complete removal of Cryptosporidium oocysts from drinking-water. Consequently, Cryptosporidium-contaminated drinking-water supplies can severely implicate both water utilities and consumers. Existing methods for the detection of Cryptosporidium in drinking-water do not discern between non-pathogenic and pathogenic species, nor between viable and non-viable oocysts. Using FluidFM, a novel force spectroscopy method employing microchannelled cantilevers for single-cell level manipulation, we assessed the size and deformability properties of two species of Cryptosporidium that pose varying levels of risk to human health. A comparison of such characteristics demonstrated the ability of FluidFM to discern between Cryptosporidium muris and Cryptosporidium parvum with 86% efficiency, whilst using a measurement throughput which exceeded 50 discrete oocysts per hour. In addition, we measured the deformability properties for untreated and temperature-inactivated oocysts of the highly infective, human pathogenic C. parvum to assess whether deformability may be a marker of viability. Our results indicate that untreated and temperature-inactivated C. parvum oocysts had overlapping but significantly different deformability distributions.     

Identification and characterization of splice variants of the human P2X7 ATP channel

The P2X7 channel is a member of the P2X family of ligand-gated ion channels which respond to ATP as the endogenous agonist. Studies suggest that P2X7 has a potentially pivotal role in inflammatory responses largely stemming from its role in mediating the release of IL-1beta in response to ATP. We report the identification of seven variants of human P2X7 which result from alternative splicing. Two of these variants (one lacking the first transmembrane domain, the second lacking the entire cytoplasmic tail) were compared to the full-length channel. Real-time PCR analysis demonstrated that both variants were expressed in various tissues and that the cytoplasmic tail deleted variant is highly expressed. Deletion of the first transmembrane domain resulted in a non-functional channel. Deletion of the cytoplasmic tail did not affect ion movement but severely affected the ability to form a large pore and to induce activation of caspases.     

Further Studies on the Ribosomal RNA Cistrons of SCIARA COPROPHILA (Diptera)

Additional experiments with homologous as well as heterologous hybridization confirmed our previous finding in Sciara coprophila that XX females have nearly twice the number of ribosomal RNA cistrons as XO males. A comparison between two different X' chromosomes revealed that only the one carrying the irradiation-induced Wavy mutation has a deletion of 70% of its ribosomal RNA cistrons as compared to the standard X. The deletion is relatively stable, and the remaining ribosomal RNA cistrons donot appear to undergo disproportionate replication or magnification as in Drosophila. Homologous hybridization experiments revealed an unusually low reiteration of ribosomal RNA cistrons in this fly, 45 gene copies per X chromosome. The question is raised as to whether such a low number of cistrons may be related to the unusual nucleolar condition encountered in the Sciaridae.     

Opt-Out Panel Testing for HIV,Hepatitis B and Hepatitis C in an Urban Emergency Department: A Pilot Study

Objectives

Studies suggest 2 per 1000 people in Dublin are living with HIV, the level above which universal screening is advised. We aimed to assess the feasibility and acceptability of a universal opt-out HIV, Hepatitis B and Hepatitis C testing programme for Emergency Department patients and to describe the incidence and prevalence of blood-borne viruses in this population.

Methods

An opt-out ED blood borne virus screening programme was piloted from March 2014 to January 2015. Patients undergoing blood sampling during routine clinical care were offered HIV 1&2 antibody/antigen assay, HBV surface antigen and HCV antibody tests. Linkage to care where necessary was co-ordinated by the study team. New diagnosis and prevalence rates were defined as the new cases per 1000 tested and number of positive tests per 1000 tested respectively.

Results

Over 45 weeks of testing, of 10,000 patient visits, 8,839 individual patient samples were available for analysis following removal of duplicates. A sustained target uptake of >50% was obtained after week 3. 97(1.09%), 44(0.49%) and 447(5.05%) HIV, Hepatitis B and Hepatitis C tests were positive respectively. Of these, 7(0.08%), 20(0.22%) and 58(0.66%) were new diagnoses of HIV, Hepatitis B and Hepatitis C respectively. The new diagnosis rate for HIV, Hepatitis B and Hepatitis C was 0.8, 2.26 and 6.5 per 1000 and study prevalence for HIV, Hepatitis B and Hepatitis C was 11.0, 5.0 and 50.5 per 1000 respectively.

Conclusions

Opt-out blood borne viral screening was feasible and acceptable in an inner-city ED. Blood borne viral infections were prevalent in this population and newly diagnosed cases were diagnosed and linked to care. These results suggest widespread blood borne viral testing in differing clinical locations with differing population demographic risks may be warranted.     

Differential responses of three Agriotes click beetle species to pheromone traps

1 Previous work had suggested that adult male click beetles (Agriotes spp.) show differential responses to species specific pheromone traps. This hypothesis was tested using mark–release–recapture methods to estimate the maximum sampling range and the effective sampling area of traps for three species. 2 Captured beetles of the species Agriotes lineatus, Agriotes obscurus and Agriotes sputator were marked to show the direction of release, the distance of the release point from the trap and the replicate. Analysis of variance showed that there were significant differences in recapture rates between species and release distances. There were no significant differences between release direction and replicates. 3 Calculated linear speeds suggested differences in movement rates in the order: A. lineatus > A. obscurus > A. sputator. There were also substantial differences between the species in the maximum sampling ranges and effective sampling areas of the traps. These placed the species in the same order. 4 The results are used to estimate the minimum cost of mass trapping programmes to prevent males from mating, giving values of €165/ha/year (A. lineatus), €247.5/ha/year (A. obscurus) and €2343/ha/year (A. sputator). 5 Implications for the use of pheromone traps in wireworm pest management are discussed. It is concluded that current U.K. recommendations based on the cumulative total catch of the three species over a sampling season can be improved by considering the spatial relationships between the adult trapping system and larval distribution. The current constraint to this is the general inability to separate wireworms into species.     

The role of zooplankton grazing in the formation of `clear water phase' in a shallow charophyte-dominated lake

In Chara-dominated shallow eutrophic Lake Prossa (Estonia), the collapse of spring phytoplankton community occurred in late May after which both primary production (PP) and phytoplankton biomass (B&pinf;) stayed at a very low level. By mid-June the Secchi depth had increased up to 2.6 m indicating the achievement of the `clear water phase', which persisted thoughout the rest of the vegetation period. The biomass of `edible' phytoplankton formed on average 53% of the total phytoplankton biomass, and the share of herbivorous zooplankton was on average 61% of the total zooplankton biomass. In spring zooplankton removed daily 27% of the total B&pinf; and 29% of PP by grazing while in summer these values rarely exceeded 5%. Zooplankton grazing was responsible for the decrease of `edible' (<31 μm) phytoplankton after its spring peak as well as for maintaining its biomass at a very low level during the whole vegetation period. Depletion of mineral forms of nitrogen and phosphorus that occurred most probably because of the development of charophytes by the end of May supported the collapse of the whole phytoplankton community and kept the water clear throughout the summer and autumn.

     

Diverse energetic effects of charge reversal mutations of poxvirus topoisomerase IB

A key aspect of the reaction mechanism of type IB topoisomerases is the controlled unwinding of DNA supercoils while the enzyme is transiently bound to one strand of the DNA duplex via a phosphotyrosyl linkage. In this complex, the mobile segment of the bound DNA downstream from the site of cleavage must rotate around the helical axis, requiring that interactions with the enzyme must break and re-form multiple times during the course of removing supercoils. A crystal structure of variola virus type IB topoisomerase (vTopo) bound to DNA shows several positively charged side chains that interact with the downstream mobile and upstream rigid segments, suggesting that these groups may play a role in catalysis, including the processive unwinding of supercoils. We have mutated three such residues, R67, K35, and K271, to Ala and Glu and determined the energetic effects of these mutations at each point along the reaction coordinate of vTopo. R67 interacts with a phosphate group in the rigid DNA segment across from the site of DNA strand cleavage. The ~30-fold damaging effects of the R67A and R67E mutations were primarily on the phosphoryl transfer step, with little effect on enzyme-DNA binding, or the processivity of supercoil unwinding. Removal of the K35 interaction shows mutational effects similar to those of R67, even though this residue interacts with the mobile segment 3 bp from the cleavage site. The two mutations of K271, which interacts with the mobile region even further from the site of covalent linkage, show significant effects not only on phosphoryl transfer but also on downstream DNA strand positioning. Moreover, supercoil unwinding measurements indicate that the K271A and K271E mutations increase the average number of supercoils that are removed during the lifetime of the covalent complex, enhancing the processivity of supercoil unwinding. These measurements support the proposal that the processivity of supercoil unwinding can be regulated by electrostatic interactions between the enzyme and the mobile DNA phosphate backbone.