Induction of immunodeficiency virus-specific immune responses in rhesus monkeys following gene gun-mediated DNA vaccination

Abstract: The Accell® gene delivery system (gene gun) was used to deliver gold particles coated with HIV-1_LAI and SIV_mac239 expression constructs into the epidermis of rhesus macaques, resulting in the elicitation of env- and gag-specific humoral responses. One microgram of vector DNA per dose was sufficient to induce immune responses in monkeys using SIV_mac239 gp160 and gp120 vectors driven by the CMV-intron A promoter. Several parameters, including the identity of the vector, the length of the rest period between immunizations, the number of immunizations, and the amount of DNA per immunization, are all important in designing an optimal DNA immunization regimen. In addition, gene gun-based DNA immunization using low efficiency expression vectors is an effective means of priming for the induction of vigorous antibody responses in macaques following boosting with recombinant subunits.     

No longer locally extinct? Tracing the origins of a lion (<Emphasis Type="Italic">Panthera leo</Emphasis>) living in Gabon

Lions (Panthera leo) are of particular conservation concern due to evidence of recent, widespread population declines in what has hitherto been seen as a common species, robust to anthropogenic disturbance. Here we use non-invasive methods to recover complete mitochondrial genomes from single hair samples collected in the field in order to explore the identity of the Gabonese Plateaux Batéké lion. Comparison of the mitogenomes against a comprehensive dataset of African lion sequences that includes relevant geographically proximate lion populations from both contemporary and ancient sources, enabled us to identify the Plateaux Batéké lion as a close maternal relative to now extirpated populations found in Gabon and nearby Congo during the twentieth century, and to extant populations of Southern Africa. Our study demonstrates the relevance of ancient DNA methods to field conservation work, and the ability of trace field samples to provide copious genetic information about free-ranging animals.     

Species Richness and Patterns of Invasion in Plants, Birds, and Fishes in the United States*

We quantified broad-scale patterns of species richness and species density (mean # species/km²) for native and non-indigenous plants, birds, and fishes in the continental USA and Hawaii. We hypothesized that the species density of native and non-indigenous taxa would generally decrease in northern latitudes and higher elevations following declines in potential evapotranspiration, mean temperature, and precipitation. County data on plants (n = 3004 counties) and birds (n=3074 counties), and drainage (6 HUC) data on fishes (n = 328 drainages) showed that the densities of native and non-indigenous species were strongly positively correlated for plant species (r = 0.86, P < 0.0001), bird species (r = 0.93, P<0.0001), and fish species (r = 0.41, P<0.0001). Multiple regression models showed that the densities of native plant and bird species could be strongly predicted (adj. R² = 0.66 in both models) at county levels, but fish species densities were less predictable at drainage levels (adj. R² = 0.31, P<0.0001). Similarly, non-indigenous plant and bird species densities were strongly predictable (adj. R² = 0.84 and 0.91 respectively), but non-indigenous fish species density was less predictable (adj. R² = 0.38). County level hotspots of native and non-indigenous plants, birds, and fishes were located in low elevation areas close to the coast with high precipitation and productivity (vegetation carbon). We show that (1) native species richness can be moderately well predicted with abiotic factors; (2) human populations have tended to settle in areas rich in native species; and (3) the richness and density of non-indigenous plant, bird, and fish species can be accurately predicted from biotic and abiotic factors largely because they are positively correlated to native species densities. We conclude that while humans facilitate the initial establishment, invasions of non-indigenous species, the spread and subsequent distributions of non-indigenous species may be controlled largely by environmental factors. The U.S. Government’s right to retain a non-exclusive, royalty-free licence in and to any copyright is acknowledged.     

PCR gut analysis reveals that Tenuiphantes tenuis (Araneae: Linyphiidae) is a potentially significant predator of Argentine stem weevil,Listronotus bonariensis (Coleoptera: Curculionidae), in New Zealand pastures

Polymerase chain reaction (PCR) gut analysis was conducted on specimens of the introduced spider Tenuiphantes tenuis collected from dairy pasture in Canterbury, New Zealand. PCR primers were specifically designed to amplify a fragment of the mitochondrial gene cytochrome c oxidase subunit 1 (COI) from Listronotus bonariensis and revealed that this major pasture pest species is consumed in the field by T. tenuis. The field predation rate of L. bonariensis by T. tenuis was estimated from our PCR results together with published data on the degradation of DNA and the density of T. tenuis in Canterbury pastures. We found that T. tenuis is a potentially significant predator of L. bonariensis in New Zealand pastures.     

Reovirus removal and inactivation by slow-rate sand filtration.

Laboratory column studies were conducted at the Utah Water Research Laboratory, Logan, Utah, to evaluate reovirus removal from drinking water supplies by slow-rate sand filtration (SSF). Columns, constructed to simulate a full-scale SSF field operation, were inoculated with reovirus at ca. 1,000-times-greater concentrations than those typically found in domestic sewage. Reovirus removal and inactivation were investigated as functions of filter maturity and other filter sand characteristics. Reovirus removal studies demonstrated that the SSF process is capable of reducing reovirus in influent water by a minimum of 4 log concentration units under certain conditions of water quality, flow rate, and sand bed construction. Infectious reovirus was not detected in effluent samples from any of the sand beds studied, after inoculation of the SSF columns; therefore, removal efficiencies were not affected significantly by characteristics, including age, of the two filter sands evaluated. Studies conducted with radioactively labeled reovirus demonstrated that reovirus removed from influent water was distributed throughout the entire length of the filter beds. Concentrations of reovirus in the filter sands decreased with increasing bed depth. The greatest removal occurred in the top few centimeters of all sand beds. No infectious reovirus could be detected in clean or mature sand bed media, indicating that reoviruses were inactivated in the filter.     

Alternate splicing and expression of the glutamate transporter EAAT5 in the rat retina

Excitatory amino acid transporter 5 (EAAT5) is an unusual glutamate transporter that is expressed in the retina, where it is localised to two populations of glutamatergic neurons, namely the bipolar neurons and photoreceptors. EAAT5 exhibits two distinct properties, acting both as a slow glutamate transporter and as a glutamate-gated inhibitory receptor. The latter property is attributable to a co-associated chloride conductance. EAAT5 has previously been thought to exist only as a full-length form. We now demonstrate by PCR cloning and sequencing, the presence of five novel splice variant forms of EAAT5 which skip either partial or complete exons in the rat retina. Furthermore, we demonstrate that each of these variants is expressed at the protein level as assessed by Western blotting using splice-specific antibodies that we have generated. We conclude that EAAT5 exists in multiple spliced forms, and propose, based upon retention or absence of key structural features, that these variant forms may potentially exhibit distinct properties relative to the originally described form of EAAT5.     

Progressive Injury in Chronic Multiple Sclerosis Lesions Is Gender-Specific: A DTI Study

Objective

To evaluate the longitudinal integrity of white matter tracts in patients with relapsing remitting multiple sclerosis (RRMS) as determined by changes in diffusivity indices of lesional and non-lesional white matter in the optic radiation over 12 months.

Methods

The optic radiation (OR) was identified in sixty RRMS patients using probabilistic tractography. MS lesions were segmented on FLAIR T2 images and a lesion mask was intersected with the co-registered OR. Lesions within the OR were identified in 39 patients. Voxel-based analysis of axial diffusivity (AD) and radial diffusivity (RD) within OR lesions and non-lesional normal appearing white matter (NAWM) was performed at baseline and 12 months in 34 patients (five patients excluded due to new OR lesions).

Results

Both RD and AD demonstrated much higher values within the lesions compared with non-lesional NAWM. There was a significant (p<0.001) increase of lesional AD and RD during the follow-up period. This increase, however, was driven almost entirely by the male cohort, in which a significantly greater change in both AD (M-2.7%, F-0.9%) and RD (M-4.6%, F-0.7%) was observed during the follow-up period. Non-lesional NAWM also demonstrated an increase in both AD and RD, albeit on a much lesser scale (1.0% and 0.6% respectively). In contradistinction to lesions, the diffusivity change in non-lesional NAWM was similar between sexes.

Conclusions

The evolution of AD and RD in chronic MS lesions over 12 months suggests ongoing inflammatory demyelinating activity accompanied by axonal loss. In addition, our findings are consistent with the recently observed trend of more rapid clinical progression in males and establish a potential in vivo biomarker of gender dichotomy by demonstrating a significantly faster rate of microstructural change in the chronic lesions of male patients with MS.     

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.