Detection of Hepatitis B Virus (HBV) DNA at femtomolar concentrations using a silica nanoparticle-enhanced microcantilever sensor

We report Hepatitis B Virus (HBV) DNA detection using a silica nanoparticle-enhanced dynamic microcantilever biosensor. A 243-mer nucleotide of HBV DNA precore/core region was used as the target DNA. For this assay, the capture probe on the microcantilever surface and the detection probe conjugated with silica nanoparticles were designed specifically for the target DNA. For efficient detection of the HBV target DNA using silica nanoparticle-enhanced DNA assay, the size of silica nanoparticles and the dimension of microcantilever were optimized by directly binding the silica nanoparticles through DNA hybridization. In addition, the correlation between the applied nanoparticle concentrations and the resonant frequency shifts of the microcantilever was discussed clearly to validate the quantitative relationship between mass loading and resonant frequency shift.HBV target DNAs of 23.1 fM to 2.31 nM which were obtained from the PCR product were detected using a silica nanoparticle-enhanced microcantilever. The HBV target DNA of 243-mer was detected up to the picomolar (pM) level without nanoparticle enhancement and up to the femtomolar (fM) level using a nanoparticle-based signal amplification process. In the above two cases, the resonant frequency shifts were found to be linearly correlated with the concentrations of HBV target DNAs. We believe that this linearity originated mainly from an increase in mass that resulted from binding between the probe DNA and HBV PCR product, and between HBV PCR product and silica nanoparticles for the signal enhancement, even though there is another potential factor such as the spring constant change that may have influenced on the resonant frequency of the microcantilever.     

GDSL lipase-like 1 regulates systemic resistance associated with ethylene signaling in Arabidopsis

Systemic resistance is induced by necrotizing pathogenic microbes and non-pathogenic rhizobacteria and confers protection against a broad range of pathogens. Here we show that Arabidopsis GDSL LIPASE-LIKE 1 (GLIP1) plays an important role in plant immunity, eliciting both local and systemic resistance in plants. GLIP1 functions independently of salicylic acid but requires ethylene signaling. Enhancement of GLIP1 expression in plants increases resistance to pathogens including Alternaria brassicicola , Erwinia carotovora and Pseudomonas syringae , and limits their growth at the infection site. Furthermore, local treatment with GLIP1 proteins is sufficient for the activation of systemic resistance, inducing both resistance gene expression and pathogen resistance in systemic leaves. The PDF1.2 -inducing activity accumulates in petiole exudates in a GLIP1-dependent manner and is fractionated in the size range of less than 10 kDa as determined by size exclusion chromatography. Our results demonstrate that GLIP1-elicited systemic resistance is dependent on ethylene signaling and provide evidence that GLIP1 may mediate the production of a systemic signaling molecule(s).     

Isolation of the protein tyrosine phosphatase 1B inhibitory metabolite from the marine-derived fungus Cosmospora sp. SF-5060

In the course of bioassay-guided study on the EtOAc extract of a culture broth of the marine-derived fungus Cosmospora sp. SF-5060, aquastatin A (1) was isolated as a protein tyrosine phosphatase 1B (PTP1B) inhibitory component produced by the fungus. The compound was isolated by various chromatographic methods, and the structure was determined mainly by analysis of NMR spectroscopic data. Compound 1 exhibited potent inhibitory activity against PTP1B with IC₅₀ value of 0.19 μM, and the kinetic analyses of PTP1B inhibition by compound 1 suggested that the compound is inhibiting PTP1B activity in a competitive manner. Aquastatin A (1) also showed modest but selective inhibitory activity toward PTP1B over other protein tyrosine phosphatases, such as TCPTP, SHP-2, LAR, and CD45. In addition, the result of hydrolyzing aquastatin A (1) suggested that the dihydroxypentadecyl benzoic acid moiety in the molecule is responsible for the inhibitory activity.     

Thyroglobulin may affect telomerase activity in thyroid follicular cells

Telomerase (TA) activity is known to be present in malignant tumor cells, but not in most somatic differentiated cells. TA shows relatively high activity in thyroid cancer cells, but reports vary. This fact prompted us to elucidate whether cell component inhibitors of TA in the thyroid follicles can modulate its activity. The activity of TA extracted from Hela cells was inhibited by mixing with the supernatant fraction of human thyroid tissue extract. To examine the effect of iodine, thyroid hormones (l-T3 and l-T4) and human thyroglobulin (hTg) contained in the thyroid follicles, l-T3, l-T4 and hTg were added to the TRAP assay system in vitro, using TA from Hela cells. Iodine, l-T3 and l-T4 did not affect TA activity, but hTg inhibited the TA activity in a dose-dependent manner (IC(50) of hTg: ca 0.45 microM: inhibiting concentration of hTg was from 0.15 microM to 3.0 microM). The hTg inhibition was not evident in the RT-PCR system, suggesting no effect of hTg on Taq DNA polymerase activity. The hTg inhibition of TA activity was attenuated by dNTP but not significantly by TS primer. These data suggest that hTg contained in thyroid follicular cells of various thyroid diseases may affect the TA activity measured in biopsied thyroid specimens, and that the reduction of the TA activity by hTg may induce slow progression and growth, and low grade malignancy of thyroid cancer, particularly differentiated carcinoma.     

Intra-arterial delivery of triolein emulsion increases vascular permeability in skeletal muscles of rabbits

Background  

To test the hypothesis that triolein emulsion will increase vascular permeability of skeletal muscle.     

Indole glucosinolate breakdown and its biological effects

Most species in the Brassicaceae produce one or more indole glucosinolates. In addition to the parent indol-3-ylmethylglucosinolate (IMG), other commonly encountered indole glucosinolates are 1-methoxyIMG, 4-hydroxyIMG, and 4-methoxyIMG. Upon tissue disruption, enzymatic hydrolysis of IMG produces an unstable aglucone, which reacts rapidly to form indole-3-acetonitrile and indol-3-ylmethyl isothiocyanate. The isothiocyanate, in turn, can react with water, ascorbate, glutathione, amino acids, and other plant metabolites to produce a variety of physiologically active indole compounds. Myrosinase-initiated breakdown of the substituted indole glucosinolates proceeds in a similar manner to that of IMG. Induction of indole glucosinolate production in response to biotic stress, experiments with mutant plants, and artificial diet assays suggest a significant role for indole glucosinolates in plant defense. However, some crucifer-feeding specialist herbivores recognize indole glucosinolates and their breakdown products as oviposition and/or feeding stimulants. In mammalian diets, IMG can have both beneficial and deleterious effects. Most IMG breakdown products induce the synthesis of phase 1 detoxifying enzymes, which may in some cases prevent carcinogenesis, but in other cases promote carcinogenesis. Recent advances in indole glucosinolate research have been fueled by their occurrence in the well-studied model plant Arabidopsis thaliana. Knowledge gained from genetic and biochemical experiments with A. thaliana can be applied to gain new insight into the ecological and nutritional properties of indole glucosinolates in other plant species.     

Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence

The pathogenic mechanism of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK) by Acanthamoeba has yet to be clarified. Protease has been recognized to play an important role in the pathogenesis of GAE and AK. In the present study, we have compared specific activity and cytopathic effects (CPE) of purified 33 kDa serine proteinases from Acanthamoeba strains with different degree of virulence (A. healyi OC-3A, A. lugdunensis KA/E2, and A. castellanii Neff). Trophozoites of the 3 strains revealed different degrees of CPE on human corneal epithelial (HCE) cells. The effect was remarkably reduced by adding phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor. This result indicated that PMSF-susceptible proteinase is the main component causing cytopathy to HCE cells by Acanthamoeba. The purified 33 kDa serine proteinase showed strong activity toward HCE cells and extracellular matrix proteins. The purified proteinase from OC-3A, the most virulent strain, demonstrated the highest enzyme activity compared to KA/E2, an ocular isolate, and Neff, a soil isolate. Polyclonal antibodies against the purified 33 kDa serine proteinase inhibit almost completely the proteolytic activity of culture supernatant of Acanthamoeba. In line with these results, the 33 kDa serine proteinase is suggested to play an important role in pathogenesis and to be the main component of virulence factor of Acanthamoeba.     

Cost-effectiveness of primary prophylaxis of AIDS associated cryptococcosis in Cambodia

Background

Cryptococcal infection is a frequent cause of mortality in Cambodian HIV-infected patients with CD4+ count ≤100 cells/µl. This study assessed the cost-effectiveness of three strategies for cryptococcosis prevention in HIV-infected patients.

Methods

A Markov decision tree was used to compare the following strategies at the time of HIV diagnosis: no intervention, one time systematic serum cryptococcal antigen (CRAG) screening and treatment of positive patients, and systematic primary prophylaxis with fluconazole. The trajectory of a hypothetical cohort of HIV-infected patients with CD4+ count ≤100 cells/µl initiating care was simulated over a 1-year period (cotrimoxazole initiation at enrollment; antiretroviral therapy within 3 months). Natural history and cost data (US$ 2009) were from Cambodia. Efficacy data were from international literature.

Results

In a population in which 81% of patients had a CD4+ count ≤50 cells/ µl and 19% a CD4+ count between 51–100 cells/µl, the proportion alive 1 year after enrolment was 61% (cost $ 472) with no intervention, 70% (cost $ 483) with screening, and 72% (cost $ 492) with prophylaxis. After one year of follow-up, the cost-effectiveness of screening vs. no intervention was US$ 180/life year gained (LYG). The cost-effectiveness of prophylaxis vs. screening was $ 511/LYG. The cost-effectiveness of prophylaxis vs. screening was estimated at $1538/LYG if the proportion of patients with CD4+ count ≤50 cells/µl decreased by 75%.

Conclusion

In a high endemic area of cryptococcosis and HIV infection, serum CRAG screening and prophylaxis are two cost effective strategies to prevent AIDS associated cryptococcosis in patients with CD4+ count ≤100 cells/µl, at a short-term horizon, screening being more cost-effective but less effective than prophylaxis. Systematic primary prophylaxis may be preferred in patients with CD4+ below 50 cells/µl while systematic serum CRAG screening for early targeted treatment may be preferred in patients with CD4+ between 51–100 cells/µl.     

The structural flexibility of the shank1 PDZ domain is important for its binding to different ligands

The PDZ domain of the shank protein interacts with numerous cell membrane receptors and cytosolic proteins via the loosely defined binding motif X-(Ser/Thr)-X-Φ-COOH (Φ represents hydrophobic residues) at the carboxyl terminus of its target protein. This enables shank to serve as a membrane-associated scaffold for the assembly of signaling complexes. As the list of proteins that bind to the shank PDZ domain grows, it is not immediately clear what structural element(s) mediate this domain’s target specificity or the plasticity required to bind its different targets. Here, we have determined the crystal structure of the shank1 PDZ in complex with the βPIX C-terminal pentapeptide (642–646, DETNL) at 2.3 Å resolution and modeled shank1 PDZ binding to selected pentapeptide ligands. The resulting structures revealed a large hydrophobic pocket within the PDZ domain that can accommodate a variety of ligand residues at the P(0) position. A H-bond between His735 and Ser/Thr at the P(−2) position is invariant throughout the model structures. In addition, we identified multiple PDZ domain residues that are able to form H-bonds and salt bridges with an incoming target protein. Overall, our study provides a new level of understanding of the specificity and structural plasticity of the shank PDZ domain.