A Simple Isothermal DNA Amplification Method to Screen Black Flies for Onchocerca volvulus Infection

Onchocerciasis is a debilitating neglected tropical disease caused by infection with the filarial parasite Onchocerca volvulus. Adult worms live in subcutaneous tissues and produce large numbers of microfilariae that migrate to the skin and eyes. The disease is spread by black flies of the genus Simulium following ingestion of microfilariae that develop into infective stage larvae in the insect. Currently, transmission is monitored by capture and dissection of black flies and microscopic examination of parasites, or using the polymerase chain reaction to determine the presence of parasite DNA in pools of black flies. In this study we identified a new DNA biomarker, encoding O. volvulus glutathione S-transferase 1a (OvGST1a), to detect O. volvulus infection in vector black flies. We developed an OvGST1a-based loop-mediated isothermal amplification (LAMP) assay where amplification of specific target DNA is detectable using turbidity or by a hydroxy naphthol blue color change. The results indicated that the assay is sensitive and rapid, capable of detecting DNA equivalent to less than one microfilaria within 60 minutes. The test is highly specific for the human parasite, as no cross-reaction was detected using DNA from the closely related and sympatric cattle parasite Onchocerca ochengi. The test has the potential to be developed further as a field tool for use in the surveillance of transmission before and after implementation of mass drug administration programs for onchocerciasis.     

Accelerating regrowth of temperate‐maritime forests due to environmental change

To understand how environmental changes have influenced forest productivity, stemwood biomass (B) dynamics were analyzed at 1267 permanent inventory plots, covering a combined 209 ha area of unmanaged temperate‐maritime forest in southwest British Columbia, Canada. Net stemwood production (ΔB) was derived from periodic remeasurements of B collected over a 40‐year measurement period (1959–1998) in stands ranging from 20 to 150 years old. Comparison between the integrated age response of net stemwood production, ΔB(A), and the age response of stemwood biomass, B(A), suggested a 58 ± 11% increase in ΔB between the first 40 years of the chronosequence period (1859–1898) and the measurement period. To estimate extrinsic forcing on ΔB, several different candidate models were developed to remove variation explained by intrinsic factors. All models exhibited temporal bias, with positive trends in (observed minus predicted) residual ΔB ranging between of 0.40 and 0.64% yr^?1. Applying the same methods to stemwood growth (G) indicated residual increases ranging from 0.43 and 0.67% yr^?1. Higher trend estimates corresponded with models that included site index (SI) as a predictor, which may reflect exaggeration of the age‐decline in SI tables. Choosing a model that excluded SI, suggested that ΔB increased by 0.40 ± 0.18% yr^?1, while G increased by 0.43 ± 0.12% yr^?1 over the measurement period. Residual G was significantly correlated with atmospheric carbon dioxide (CO₂), temperature (T), and climate moisture index (CMI). However, models driven with climate and CO₂, alone, could not simultaneously explain long‐term and measurement‐period trends without additional representation of indirect effects, perhaps reflecting compound interest on direct physiological responses to environmental change. Evidence of accelerating forest regrowth highlights the value of permanent inventories to detect and understand systematic changes in forest productivity caused by environmental change.     

Detection of Peptide-Based Nanoparticles in Blood Plasma by ELISA

AimsThe aim of the current study was to develop a method to detect peptide-linked nanoparticles in blood plasma.ResultsThe ELISA based method for the detection of FITC labeled peptides had a detection limit of 1 ng/mL. We were able to accurately measure peptides bound to pentafluorophenyl methacrylate nanoparticles in blood plasma of rats, and similar results were obtained by LC/MS.ConclusionsWe detected FITC-labeled peptides on pentafluorophenyl methacrylate nanoparticles after injection in vivo. This method can be extended to detect nanoparticles with different chemical compositions.     

Identification of interferon-stimulated gene 15 as an antiviral molecule during Sindbis virus infection in vivo

The innate immune response, and in particular the alpha/beta interferon (IFN-alpha/beta) system, plays a critical role in the control of viral infections. Interferons alpha and beta exert their antiviral effects through the induction of hundreds of interferon-induced (or -stimulated) genes (ISGs). While several of these ISGs have characterized antiviral functions, their actions alone do not explain all of the effects mediated by IFN-alpha/beta. To identify additional IFN-induced antiviral molecules, we utilized a recombinant chimeric Sindbis virus to express selected ISGs in IFN-alpha/beta receptor (IFN-alpha/betaR)(-/-) mice and looked for attenuation of Sindbis virus infection. Using this approach, we identified a ubiquitin homolog, interferon-stimulated gene 15 (ISG15), as having antiviral activity. ISG15 expression protected against Sindbis virus-induced lethality and decreased Sindbis virus replication in multiple organs without inhibiting the spread of virus throughout the host. We establish that, much like ubiquitin, ISG15 requires its C-terminal LRLRGG motif to form intracellular conjugates. Finally, we demonstrate that ISG15's LRLRGG motif is also required for its antiviral activity. We conclude that ISG15 can be directly antiviral.     

Isolation and Characterization of Twelve Microsatellite Loci for Rockfish (Sebastes)

We describe the first microsatellites for rockfishes in the diverse genus Sebastes. Clones containing microsatellites were isolated from the genomic library of a quillback rockfish, Sebastes maliger. Twelve microsatellites are characterized; six of these are polymorphic in quillback rockfish, and eight are polymorphic in at least one rockfish species on which they were tested. The number of alleles per variable locus ranged from 4 to 15 and averaged 6.8. The expected heterozygosities ranged from 0.38 to 0.79 and averaged 0.60 in these loci. These loci should prove valuable in studies examining species identification, population genetics, hybridization, paternity, kinship, and microsatellite evolution. Received September 8, 1998; accepted November 23, 1998.     

Detection of mRNA degradation intermediates in tissues using the 3'-end poly(A)-tailing polymerase chain reaction method

It has become increasingly clear that mRNA stability is an important determinant of mRNA abundance in virtually all organisms. Although our understanding of prokaryotic lower eukaryotic mRNA stability mechanisms has progressed considerably, little is known about mammalian mRNA stability mechanisms, particularly at the tissue and animal levels. This is due largely to the lack of suitable methods to approach the problem. In this study, we have developed and refined the 3'-end poly(A)-tailing polymerase chain reaction (PCR) method to detect degradation intermediates in vivo. Using an in vitro transcribed RNA as a template, we found that the method could be used to detect a homogeneous pool of RNA down to 0.1 ng. The addition of 10 microg of total RNA from tissues decreased the sensitivity limit to 4 ng. Detection limits of the technique were determined precisely by varying the concentrations of in vitro transcribed RNA in a constant amount of total RNA and varying the concentration of total RNA while maintaining a constant amount of in vitro transcribed RNA. Our overall results showed that the poly(A)-tailing PCR method could be used to detect specific RNA species of approximately 1000 nt in a pool of heterogeneous RNA in the range of 1 in 2500 to 1 in 10,000. To our knowledge, this is the most sensitive method to date for identifying mRNA degradation intermediates. Employing sense strand gene-specific primers in this method, we have discovered the class II and class III P-glycoprotein (Pgp) mRNA degradation intermediates in normal rat tissues. This method should serve as an additional tool to help us understand mRNA decay mechanisms in tissues and at animal levels.     

Is Cassava the Answer to African Climate Change Adaptation?

This paper examines the impacts of climate change on cassava production in Africa, and questions whether cassava can play an important role in climate change adaptation. First, we examine the impacts that climate change will likely have on cassava itself, and on other important staple food crops for Africa including maize, millets, sorghum, banana, and beans based on projections to 2030. Results indicate that cassava is actually positively impacted in many areas of Africa, with ?3.7% to +17.5% changes in climate suitability across the continent. Conversely, for other major food staples, we found that they are all projected to experience negative impacts, with the greatest impacts for beans (?16%?±?8.8), potato (?14.7?±?8.2), banana (?2.5%?±?4.9), and sorghum (?2.66%?±?6.45). We then examined the likely challenges that cassava will face from pests and diseases through the use of ecological niche modeling for cassava mosaic disease, whitefly, brown streak disease and cassava mealybug. The findings show that the geographic distribution of these pests and diseases are projected to change, with both new areas opening up and areas where the pests and diseases are likely to leave or reduce in pressure. We finish the paper by looking at the abiotic traits of priority for crop adaptation for a 2030 world, showing that greater drought tolerance could bring some benefits in all areas of Africa, and that cold tolerance in Southern Africa will continue to be a constraint for cassava despite a warmer 2030 world, hence breeding needs to keep a focus on this trait. Importantly, heat tolerance was not found to be a major priority for crop improvement in cassava in the whole of Africa, but only in localized pockets of West Africa and the Sahel. The paper concludes that cassava is potentially highly resilient to future climatic changes and could provide Africa with options for adaptation whilst other major food staples face challenges.     

Isolation of Pandemic Vibrio parahaemolyticus from UK Water and Shellfish Produce

Vibrio parahaemolyticus is a Gram-negative, halophilic bacterium found commonly in temperate and warm estuarine waters worldwide. V. parahaemolyticus is considered an emerging bacterial pathogen in Europe and has been responsible for several recent seafood-associated outbreaks. During ad hoc testing of raw shellfish produce in May 2012, pandemic group (O3:K6) V. parahaemolyticus was isolated from Pacific oysters (Crassostrea gigas), harvested in Southern England. Follow-on testing of water and shellfish, encompassing a small number geographically diverse sites, also retrieved pandemic group isolates. These strains are amongst the most northerly pandemic strains described to date and represent the first instance of pandemic V. parahaemolyticus isolated in the UK, highlighting the expanding geographical distribution of these foodborne pathogens in the environment.     

Prevalence of agglutinating antibodies to Sarcocystis neurona in skunks (Mephitis Mephitis), raccoons (Procyon lotor), and opossums (Didelphis Virginiana) from Connecticut

Equine protozoal myeloencephalitis is the most important protozoan disease of horses in North America and is usually caused by Sarcocystis neurona. Natural cases of encephalitis caused by S. neurona have been reported in skunks (Mephitis mephitis) and raccoons (Procyon lotor). Opossums (Didelphis spp.) are the only known definitive host. Sera from 24 striped skunks, 12 raccoons, and 7 opossums (D. virginiana) from Connecticut were examined for agglutinating antibodies to S. neurona using the S. neurona agglutination test (SAT) employing formalin-fixed merozoites as antigen. The SAT was validated for skunk sera using pre- and postinfection serum samples from 2 experimentally infected skunks. Of the 24 (46%) skunks 11 were positive, and all 12 raccoons were positive for S. neurona antibodies. None of the 7 opossums was positive for antibodies to S. neurona. These results suggest that exposure to sporocysts of S. neurona by intermediate hosts is high in Connecticut. The absence of antibodies in opossums collected from the same areas is most likely because of the absence of systemic infection in the definitive host.