RNAi in Haemonchus contortus: a potential method for target validation

RNA interference (RNAi) is a method for the functional analysis of specific genes, and is particularly well developed in the free-living nematode Caenorhabditis elegans. There have been several attempts to apply this method to parasitic nematodes. In a recent study undertaken in Haemonchus contortus, Geldhof and colleagues concluded that, although a mechanism for RNAi existed, the methods developed for RNAi in C. elegans had variable efficacy in this parasitic nematode. The potential benefits of RNAi are clear; however, further studies are required to characterize the mechanism present in parasitic nematodes, and to improve culture systems for these nematodes to monitor the long-term effects of RNAi. Only then could RNAi become a reliable assay of gene function.     

Intracytoplasmic trapping of influenza virus by a lipophilic derivative of aglycoristocetin

We report on a new anti-influenza virus agent, SA-19, a lipophilic glycopeptide derivative consisting of aglycoristocetin coupled to a phenylbenzyl-substituted cyclobutenedione. In Madin-Darby canine kidney cells infected with influenza A/H1N1, A/H3N2, or B virus, SA-19 displayed a 50% antivirally effective concentration of 0.60 μM and a selectivity index (ratio of cytotoxic versus antiviral concentration) of 112. SA-19 was 11-fold more potent than unsubstituted aglycoristocetin and was active in human and nonhuman cell lines. Virus yield at 72 h p.i. was reduced by 3.6 logs at 0.8 μM SA-19. In contrast to amantadine and oseltamivir, SA-19 did not select for resistance upon prolonged virus exposure. SA-19 was shown to inhibit an early postbinding step in virus replication. The compound had no effect on hemagglutinin (HA)-mediated membrane fusion in an HA-polykaryon assay and did not inhibit the low-pH-induced refolding of the HA in a tryptic digestion assay. However, a marked inhibitory effect on the transduction exerted by retroviral pseudoparticles carrying an HA or vesicular stomatitis virus glycoprotein (VSV-G) fusion protein was noted, suggesting that SA-19 targets a cellular factor with a role in influenza virus and VSV entry. Using confocal microscopy with antinucleoprotein staining, SA-19 was proven to completely prevent the influenza virus nuclear entry. This virus arrest was characterized by the formation of cytoplasmic aggregates. SA-19 appeared to disturb the endocytic uptake and trap the influenza virus in vesicles distinct from early, late, or recycling endosomes. The aglycoristocetin derivative SA-19 represents a new class of potent and broad-acting influenza virus inhibitors with potential clinical relevance.     

Genetic architecture in a marine hybrid zone: comparing outlier detection and genomic clines analysis in the bivalve Macoma balthica

The role of natural selection in speciation has received increasing attention and support in recent years. Different types of approaches have been developed that can detect genomic regions influenced by selection. Here, we address the question whether two highly different methods--F(ST) outlier analysis and admixture analysis--detect largely the same set of non-neutral genomic elements or, instead, complementary sets. We study genetic architecture in a natural secondary contact zone where extensive admixture occurs. The marine bivalves Macoma balthica rubra and M. b. balthica descend from two independent trans-Arctic invasions of the north Atlantic and hybridize extensively where they meet, for example in the Kattegat-Danish Straits-Baltic Sea region. The Kattegat-Danish Straits region forms a steep salinity cline and is the only entrance to the recently (ca. 8000 years ago) established brackish water basin the Baltic Sea. Salinity along the contact zone drops from 30‰ (Skagerrak, M.b.rubra) to 3‰ (Baltic, M.b.balthica). Both outlier analysis and genomic clines analysis suggest that large parts of the genome are influenced by non-neutral effects. Contrasting samples from well outside the hybrid zone, outlier analysis detects 16 of 84 amplified fragment length polymorphism markers as significant F(ST) outliers. Genomic clines analysis detects 31 of 84 markers as non-neutral inside the hybrid zone. Remarkably, only three markers are detected by both methods. We conclude that the two methods together identify a suite of markers that are under the influence of non-neutral effects.     

Development of new plasmid DNA vaccine vectors with R1-based replicons

ABSTRACT: BACKGROUND: There has been renewed interest in biopharmaceuticals based on plasmid DNA (pDNA) in recent years due to the approval of several veterinary DNA vaccines, on-going clinical trials of human pDNA-based therapies, and significant advances in adjuvants and delivery vehicles that have helped overcome earlier efficacy deficits. With this interest comes the need for high-yield, cost-effective manufacturing processes. To this end, vector engineering is one promising strategy to improve plasmid production. RESULTS: In this work, we have constructed a new DNA vaccine vector, pDMB02-GFP, containing the runaway R1 origin of replication. The runaway replication phenotype should result in plasmid copy number amplification after a temperature shift from 30degreesC to 42degreesC. However, using Escherichia coli DH5alpha as a host, we observed that the highest yields of pDMB02-GFP were achieved during constant-temperature culture at 30degreesC, with a maximum yield of approximately 19 mg pDNA/g DCW being observed. By measuring mRNA and protein levels of the R1 replication initiator protein, RepA, we determined that RepA may be limiting pDMB02-GFP yield at 42degreesC. A mutant plasmid, pDMB-ATG, was constructed by changing the repA start codon from the sub-optimal GTG to ATG. In cultures of DH5alpha[pDMB-ATG], temperature-induced plasmid amplification was more dramatic than that observed with pDMB02-GFP, and RepA protein was detectable for several hours longer than in cultures of pDMB02-GFP at 42degreesC. CONCLUSIONS: Overall, we have demonstrated that R1-based plasmids can produce high yields of high-quality pDNA without the need for a temperature shift, and have laid the groundwork for further investigation of this class of vectors in the context of plasmid DNA production.     

Plasminogen activator inhibitor type I may contribute to transient, non-specific changes in immunity in the subacute phase of murine tuberculosis

Tuberculosis, caused by Mycobacterium (M.) tuberculosis, is a devastating infectious disease causing many deaths worldwide. Non-specific host defense mechanisms such as the coagulation and fibrinolytic system may give insight in possible new therapeutic targets. Plasminogen activator inhibitor type-1 (PAI-1), an important regulator of inflammation and fibrinolysis, might be of interest as tuberculosis patients have elevated plasma levels of PAI-1. In this study we set out to investigate the role of PAI-1 during tuberculosis in vivo. Wildtype (WT) and PAI-1 deficient (PAI-1^?/?) mice were intranasally infected with M. tuberculosis H37rv and sacrificed after 2, 5 and 29 weeks. Five weeks post-infection, bacterial loads in lungs of PAI-1^?/? mice were significantly higher compared to WT mice, while no differences were seen 2 and 29 weeks post-infection. At two weeks post-infection increased influx of macrophages and lymphocytes was observed. PAI-1 deficiency was associated with a reduced cytokine response in the lungs; however, upon stimulation with tuberculin purified protein derivative (PPD), PAI-1^?/? splenocytes released increased levels of IFN-γ compared to WT. No clear differences were found between PAI-1^?/? and WT mice at 29 weeks after infection. In conclusion, these data suggest that PAI-1 contributes to transient, non-specific changes in immunity during the early phase of murine tuberculosis.     

Breast cancer risk and 6q22.33: combined results from Breast Cancer Association Consortium and Consortium of Investigators on Modifiers of BRCA1/2

Recently, a locus on chromosome 6q22.33 (rs2180341) was reported to be associated with increased breast cancer risk in the Ashkenazi Jewish (AJ) population, and this association was also observed in populations of non-AJ European ancestry. In the present study, we performed a large replication analysis of rs2180341 using data from 31,428 invasive breast cancer cases and 34,700 controls collected from 25 studies in the Breast Cancer Association Consortium (BCAC). In addition, we evaluated whether rs2180341 modifies breast cancer risk in 3,361 BRCA1 and 2,020 BRCA2 carriers from 11 centers in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Based on the BCAC data from women of European ancestry, we found evidence for a weak association with breast cancer risk for rs2180341 (per-allele odds ratio (OR)?=?1.03, 95% CI 1.00-1.06, p?=?0.023). There was evidence for heterogeneity in the ORs among studies (I(2)?=?49.3%; p?=?<0.004). In CIMBA, we observed an inverse association with the minor allele of rs2180341 and breast cancer risk in BRCA1 mutation carriers (per-allele OR?=?0.89, 95%CI 0.80-1.00, p?=?0.048), indicating a potential protective effect of this allele. These data suggest that that 6q22.33 confers a weak effect on breast cancer risk.     

Site-selective glycosylation of hemoglobin on Cys beta93

In this work, we describe the synthesis and characterization of a novel glycosylated hemoglobin (Hb) with high oxygen affinity as a potential Hb-based oxygen carrier. Site-selective glycosylation of bovine Hb was achieved by conjugating a lactose derivative to Cys 93 on the beta subunit of Hb. LC-MS analysis indicates that the reaction was quantitative, with no unmodified Hb present in the reaction product. The glycosylation site was identified by chymotrypsin digestion of the glycosylated bovine Hb followed with LC-MS/MS and from the X-ray crystal structure of the glycosylated Hb. The chemical conjugation of the lactose derivative at Cys beta93 yields an oxygen carrier with a high oxygen affinity (P(50) of 4.94 mmHg) and low cooperativity coefficient (n) of 1.20. Asymmetric flow field-flow fractionation (AFFFF) coupled with multiangle static light scattering (MASLS) was used to measure the absolute molecular weight of the glycosylated Hb. AFFFF-MASLS analysis indicates that glycosylation of Hb significantly altered the alpha(2)beta(2)-alphabeta equilibrium compared to native Hb. Subsequent X-ray analysis of the glycosylated Hb crystal showed that the covalently linked lactose derivative is sandwiched between the beta(1) and alpha(2) (and hence by symmetry the beta(2) and alpha(1)) subunits of the tetramer, and the interaction between the saccharide and amino acid residues located at the interface is apparently stabilized by hydrogen bonding interactions. The resultant structural analysis of the glycosylated Hb helps to explain the shift in the alpha(2)beta(2)-alphabeta equilibrium in terms of the hydrogen bonding interactions at the beta(1)alpha(2)/beta(2)alpha(1) interface. Taken together, all of these results indicate that it is feasible to site-specifically glycosylate Hb. This work has great potential in developing an oxygen carrier with defined chemistry that can target oxygen delivery to low pO(2) tissues and organs.     

An information-theoretic analysis of genetics, gender and age in cancer patients

Germline genetics, gender and hormonal-signaling pathways are all well described modifiers of cancer risk and progression. Although an improved understanding of how germline genetic variants interact with other cancer risk factors may allow better prevention and treatment of human cancer, measuring and quantifying these interactions is challenging. In other areas of research, Information Theory has been used to quantitatively describe similar multivariate interactions. We implemented a novel information-theoretic analysis to measure the joint effect of a high frequency germline genetic variant of the p53 tumor suppressor pathway (MDM2 SNP309 T/G) and gender on clinical cancer phenotypes. This analysis quantitatively describes synergistic interactions among gender, the MDM2 SNP309 locus, and the age of onset of tumorigenesis in p53 mutation carriers. These results offer a molecular and genetic basis for the observed sexual dimorphism of cancer risk in p53 mutation carriers and a model is proposed that suggests a novel cancer prevention strategy for p53 mutation carriers.     

Biochemical evaluation of bioelectricity production process from anaerobic wastewater treatment in a single chambered microbial fuel cell (MFC) employing glass wool membrane

Biochemical functioning of single chambered microbial fuel cell (MFC) using glass wool as proton exchange membrane (PEM) operated with selectively enriched acidogenic mixed culture was evaluated in terms of bioelectricity production and wastewater treatment. Performance of MFC was studied at two different organic/substrate loading rates (OLR) (2.64 and 3.54 kg COD/m(3)) and operating pH 6 and 7 using non-coated plain graphite electrodes (mediatorless anode; air cathode). Applied OLR in association with operating pH showed marked influence on the power output and substrate degradation efficiency. Higher current density was observed at acidophilic conditions [pH 6; 98.13 mA/m(2) (2.64 kg COD/m(3)-day; 100 Omega) and 111.29 mA/m(2) (3.54 kg COD/m(3)-day; 100 Omega)] rather than neutral conditions [pH 7; 100.52 mA/m(2) (2.64 kg COD/m(3)-day; 100 Omega) and 98.13 mA/m(2) (3.54 kg COD/m(3)-day; 100 Omega)]. On the contrary, effective substrate degradation was observed at neutral pH. MFC performance was evaluated employing polarization curve, impedance analysis, cell potential, Coulombic efficiency and bioprocess monitoring. Sustainable power yield was calculated at stable cell potential.