Identification, cloning, and functional expression of three glutathione transferase genes from Aspergillus fumigatus

Analysis of the genome of the human pathogen, Aspergillus fumigatus, revealed the presence of several putative glutathione transferase (GST) open reading frames. Three A. fumigatus GST genes, termed gstA, B, and C, were cloned and recombinant proteins expressed in Escherichia coli. Functional analysis of recombinant gstA-C confirms that the enzymes exhibit GST activity and glutathione peroxidase activity. RT-PCR confirmed low basal expression of gstA and gstC which was markedly up-regulated (at least 4x-10x) in the presence of either H2O2 or 1-chloro-2,4-dinitrobenzene (CDNB). GstB expression was only observed in the presence of CDNB. These results demonstrate for the first time the existence of three functional GSTs in A. fumigatus and strongly suggest a role for these enzymes in the response of the organism to both oxidative stress and xenobiotic presence.     

Folate receptor-targeted aggregation-enhanced near-IR emitting silica nanoprobe for one-photon in vivo and two-photon ex vivo fluorescence bioimaging

A two-photon absorbing (2PA) and aggregation-enhanced near-infrared (NIR) emitting pyran derivative, encapsulated in and stabilized by silica nanoparticles (SiNPs), is reported as a nanoprobe for two-photon fluorescence microscopy (2PFM) bioimaging that overcomes the fluorescence quenching associated with high chromophore loading. The new SiNP probe exhibited aggregate-enhanced emission producing nearly twice as strong a signal as the unaggregated dye, a 3-fold increase in two-photon absorption relative to the DFP in solution, and approximately 4-fold increase in photostability. The surface of the nanoparticles was functionalized with a folic acid (FA) derivative for folate-mediated delivery of the nanoprobe for 2PFM bioimaging. Surface modification of SiNPs with the FA derivative was supported by zeta potential variation and (1)H NMR spectral characterization of the SiNPs as a function of surface modification. In vitro studies using HeLa cells expressing a folate receptor (FR) indicated specific cellular uptake of the functionalized nanoparticles. The nanoprobe was demonstrated for FR-targeted one-photon in vivo imaging of HeLa tumor xenograft in mice upon intravenous injection of the probe. The FR-targeting nanoprobe not only exhibited highly selective tumor targeting but also readily extravasated from tumor vessels, penetrated into the tumor parenchyma, and was internalized by the tumor cells. Two-photon fluorescence microscopy bioimaging provided three-dimensional (3D) cellular-level resolution imaging up to 350 μm deep in the HeLa tumor.     

Interspecific hybridization contributes to high genetic diversity and apparent effective population size in an endemic population of mottled ducks (Anas fulvigula maculosa)

Under drift-mutation equilibrium, genetic diversity is expected to be correlated with effective population size (N _e ). Changes in population size and gene flow are two important processes that can cause populations to deviate from this expected relationship. In this study, we used DNA sequences from six independent loci to examine the influence of these processes on standing genetic diversity in endemic mottled ducks (Anas fulvigula) and geographically widespread mallards (A. platyrhynchos), two species known to hybridize. Mottled ducks have an estimated census size that is about two orders-of-magnitude smaller than that of mallards, yet these two species have similar levels of genetic diversity, especially at nuclear DNA. Coalescent analyses suggest that a population expansion in the mallard at least partly explains this discrepancy, but the mottled duck harbors higher genetic diversity and apparent N _e than expected for its census size even after accounting for a population decline. Incorporating gene flow into the model, however, reduced the estimated N _e of mottled ducks to 33 % of the equilibrium N _e and yielded an estimated N _e consistent with census size. We also examined the utility of these loci to distinguish among mallards, mottled ducks, and their hybrids. Most putatively pure individuals were correctly assigned to species, but the power for detecting hybrids was low. Although hybridization with mallards potentially poses a conservation threat to mottled ducks by creating a risk of extinction by hybridization, introgression of mallard alleles has helped maintain high genetic diversity in mottled ducks and might be important for the adaptability and survival of this species.     

Influence of harmonic radar tag attachment on nymphal Halyomorpha halys mobility,survivorship, and detectability

The brown marmorated stink bug, Halyomorpha halys (Stål) (Hemiptera: Pentatomidae), is a polyphagous invasive insect and currently one of the most threatening agricultural pests in the USA and globally. Nymphs are highly mobile, moving among host plants, and causing significant damage. Thus, understanding dispersal biology for all life stages is critical for the development of reliable monitoring and management programs. Here, we evaluated the influence of harmonic radar as a tool to study dispersal ecology of nymphal H. halys; we measured the impact of glues and tag attachment on survivorship and mobility in the laboratory and validated in the field that tagged and released nymphs could be tracked on baited and unbaited host and non‐host plants using harmonic radar. In the laboratory, four glues were evaluated for attaching harmonic radar tags securely to nymphs, and survivorship with attached tags was measured. There were no significant differences in survivorship or vertical and horizontal movement among nymphs with tags affixed with the glue treatments compared with the untagged control. Based on numerically greater survivorship of nymphs with tags affixed with Loctite glass glue, a field validation study of tagged nymphs released in host (apple tree) and non‐host (mowed grass) with or without H. halys pheromonal stimuli present revealed that nymphs could be successfully relocated using harmonic radar after 48 h. Among treatments, 83% of nymphs remained in baited and unbaited apple trees, 50% of nymphs remained in baited mowed grass plots, and in unbaited mowed grass plots, 17% of fifth instars, and 0% of fourth instars were retained. The absence of negative effects on mobility, survivorship, and field tracking validates that harmonic radar can be used to study dispersal ecology of nymphal H. halys.     

HMGB1 Mediates Cognitive Impairment in Sepsis Survivors

Severe sepsis, a syndrome that complicates infection and injury, affects 750,000 annually in the United States. The acute mortality rate is approximately 30%, but, strikingly, sepsis survivors have a significant disability burden: up to 25% of survivors are cognitively and physically impaired. To investigate the mechanisms underlying persistent cognitive impairment in sepsis survivors, here we developed a murine model of severe sepsis survivors following cecal ligation and puncture (CLP) to study cognitive impairments. We observed that serum levels of high mobility group box 1 (HMGB1), a critical mediator of acute sepsis pathophysiology, are increased in sepsis survivors. Significantly, these levels remain elevated for at least 4 wks after CLP. Sepsis survivors develop significant, persistent impairments in learning and memory, and anatomic changes in the hippocampus associated with a loss of synaptic plasticity. Administration of neutralizing anti-HMGB1 antibody to survivors, beginning 1 wk after onset of peritonitis, significantly improved memory impairments and brain pathology. Administration of recombinant HMGB1 to na?ve mice recapitulated the memory impairments. Together, these findings indicate that elevated HMGB1 levels mediate cognitive decline in sepsis survivors, and suggest that it may be possible to prevent or reverse cognitive impairments in sepsis survivors by administration of anti-HMGB1 antibodies.     

A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins

A selective protonation strategy is described that uses [3-2H] 13C -ketoisovalerate to introduce (1H- methyl)-leucine and (1H- methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of 100 mg/L -ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] -ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-1 methyl)-isoleucine (>90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.     

A functional gene array for detection of bacterial virulence elements

Emerging known and unknown pathogens create profound threats to public health. Platforms for rapid detection and characterization of microbial agents are critically needed to prevent and respond to disease outbreaks. Available detection technologies cannot provide broad functional information about known or novel organisms. As a step toward developing such a system, we have produced and tested a series of high-density functional gene arrays to detect elements of virulence and antibiotic resistance mechanisms. Our first generation array targets genes from Escherichia coli strains K12 and CFT073, Enterococcus faecalis and Staphylococcus aureus. We determined optimal probe design parameters for gene family detection and discrimination. When tested with organisms at varying phylogenetic distances from the four target strains, the array detected orthologs for the majority of targeted gene families present in bacteria belonging to the same taxonomic family. In combination with whole-genome amplification, the array detects femtogram concentrations of purified DNA, either spiked in to an aerosol sample background, or in combinations from one or more of the four target organisms. This is the first report of a high density NimbleGen microarray system targeting microbial antibiotic resistance and virulence mechanisms. By targeting virulence gene families as well as genes unique to specific biothreat agents, these arrays will provide important data about the pathogenic potential and drug resistance profiles of unknown organisms in environmental samples.