Intron-derived small RNAs for silencing viral RNAs in mosquito cells

Aedes aegypti and Ae. albopictus are the main vectors of mosquito-borne viruses of medical and veterinary significance. Many of these viruses have RNA genomes. Exogenously provided, e.g. transgene encoded, small RNAs could be used to inhibit virus replication, breaking the transmission cycle. We tested, in Ae. aegypti and Ae. albopictus cell lines, reporter-based strategies for assessing the ability of two types of small RNAs to inhibit a chikungunya virus (CHIKV) derived target. Both types of small RNAs use a Drosophila melanogaster pre-miRNA-1 based hairpin for their expression, either with perfect base-pairing in the stem region (shRNA-like) or containing two mismatches (miRNA-like). The pre-miRNA-1 stem loop structure was encoded within an intron; this allows co-expression of one or more proteins, e.g. a fluorescent protein marker tracking the temporal and spatial expression of the small RNAs in vivo. Three reporter-based systems were used to assess the relative silencing efficiency of ten shRNA-like siRNAs and corresponding miRNA-like designs. Two systems used a luciferase reporter RNA with CHIKV RNA inserted either in the coding sequence or within the 3’ UTR. A third reporter used a CHIKV derived split replication system. All three reporters demonstrated that while silencing could be achieved with both miRNA-like and shRNA-like designs, the latter were substantially more effective. Dcr-2 was required for the shRNA-like siRNAs as demonstrated by loss of inhibition of the reporters in Dcr-2 deficient cell lines. These positive results in cell culture are encouraging for the potential use of this pre-miRNA-1-based system in transgenic mosquitoes.     

Optimization of a series of quinazolinone-derived antagonists of CXCR3

The evaluation of the CXCR3 antagonist AMG 487 in clinic trials was complicated due to the formation of an active metabolite. In this Letter, we will discuss the further optimization of the quinazolinone series that led to the discovery of compounds devoid of the formation of the active metabolite that was seen with AMG 487. In addition, these compounds also feature increased potency and good pharmacokinetic properties. We will also discuss the efficacy of the lead compound 34 in a mouse model of cellular recruitment induced by bleomycin.     

Allele-Specific PCR with Competitive Probe Blocking for Sensitive and Specific Detection of BRAF V600E in Thyroid Fine-Needle Aspiration Specimens

Objective: To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR). Study Design: Tumor-enriched DNA was extracted from FNA smears, formalin-fixed paraffin-embedded (FFPE) sections, or NR specimens from 37 patients with confirmed papillary thyroid carcinoma or benign findings. An allele-specific primer selectively amplified the 1799 T>A BRAF mutation while simultaneously blocking amplification of wild-type (WT) BRAF with an unlabeled probe during PCR. Mutation detection was accomplished by melting analysis of the probe. Results: Allele-specific/blocking probe PCR confirmed the BRAF mutation status for 20 of 24 paired FNA/FFPE samples previously tested by fluorescent probe real-time PCR. For the other 4 cases, the sensitive PCR method detected the BRAF mutation in all paired FNA/FFPE samples. Previously, the mutation had been detected in only the FFPE samples. The BRAF mutation was also detected in some NR specimens. Conclusion: Treatment of patients with thyroid nodules is guided by FNA biopsy, which can be scantly cellular, necessitating a sensitive test that can detect low levels of BRAF V600E mutation in a WT background. We report increased detection of BRAF V600E in FNA specimens using allele-specific/blocking probe PCR, which has an analytical sensitivity of 0.01%.     

Myeloid-derived suppressor cells are associated with disease progression and decreased overall survival in advanced-stage melanoma patients

Myeloid-derived suppressor cells are increased in the peripheral blood of advanced-stage cancer patients; however, no studies have shown a correlation of these immunosuppressive cells with clinical outcomes in melanoma patients. We characterized the frequency and suppressive function of multiple subsets of myeloid-derived suppressor cells in the peripheral blood of 34 patients with Stage IV melanoma, 20 patients with Stage I melanoma, and 15 healthy donors. The frequency of CD14⁺ MDSCs (Lin^? CD11b⁺ HLA-DR^? CD14⁺ CD33⁺) and CD14^? MDSCs (Lin^? CD11b⁺ HLA-DR^? CD14^? CD33⁺) was increased in the peripheral blood of Stage IV melanoma patients relative to healthy donors. The frequency of CD14⁺ and CD14^? MDSCs correlated with each other and with the increased frequency of regulatory T cells, but not with classically defined monocytes. CD14^? MDSCs isolated from the peripheral blood of Stage IV melanoma patients suppressed T cell activation more than those isolated from healthy donors, and the frequency of these cells correlated with disease progression and decreased overall survival. Our study provides the first evidence that the frequency of CD14^? MDSCs negatively correlates with clinical outcomes in advanced-stage melanoma patients. These data indicate that suppressive MDSCs should be considered as targets for future immunotherapies.     

Rewiring bacteria, two components at a time

In this issue, Skerker et al. (2008) present a rational method for rewiring the protein-protein interactions and output responses of prokaryotic two-component signal transduction systems. This work has important implications for understanding the specificity of protein interactions and for designing protein-based synthetic signaling cascades.     

Remodeling of synaptic actin induced by photoconductive stimulation.

Use-dependent synapse remodeling is thought to provide a cellular mechanism for encoding durable memories, yet whether activity triggers an actual structural change has remained controversial. We use photoconductive stimulation to demonstrate activity-dependent morphological synaptic plasticity by video imaging of GFP-actin at individual synapses. A single tetanus transiently moves presynaptic actin toward and postsynaptic actin away from the synaptic junction. Repetitive spaced tetani induce glutamate receptor-dependent stable restructuring of synapses. Presynaptic actin redistributes and forms new puncta that label for an active synapse marker FM5-95 within 2 hr. Postsynaptic actin sprouts projections toward the new presynaptic actin puncta, resembling the axon-dendrite interaction during synaptogenesis. Our results indicate that activity-dependent presynaptic structural plasticity facilitates the formation of new active presynaptic terminals.     

Soil Carbon Turnover Measurement by Physical Fractionation at a Forest-to-Pasture Chronosequence in the Brazilian Amazon

The effect of conversion from forest-to-pasture upon soil carbon stocks has been intensively discussed, but few studies focus on how this land-use change affects carbon (C) distribution across soil fractions in the Amazon basin. We investigated this in the 20 cm depth along a chronosequence of sites from native forest to three successively older pastures. We performed a physicochemical fractionation of bulk soil samples to better understand the mechanisms by which soil C is stabilized and evaluate the contribution of each C fraction to total soil C. Additionally, we used a two-pool model to estimate the mean residence time (MRT) for the slow and active pool C in each fraction. Soil C increased with conversion from forest-to-pasture in the particulate organic matter (>250 μm), microaggregate (53–250 μm), and d-clay (<2 μm) fractions. The microaggregate comprised the highest soil C content after the conversion from forest-to-pasture. The C content of the d-silt fraction decreased with time since conversion to pasture. Forest-derived C remained in all fractions with the highest concentration in the finest fractions, with the largest proportion of forest-derived soil C associated with clay minerals. Results from this work indicate that microaggregate formation is sensitive to changes in management and might serve as an indicator for management-induced soil carbon changes, and the soil C changes in the fractions are dependent on soil texture.     

Calmodulin in porcine malignant hyperpyrexia

The anaesthetic complication malignant hyperpyrexia (MH) is due to an elevation of the myoplasmic Ca2+ concentration. Examination of calmodulin isolated from MH susceptible swine suggests that the disorder in calcium regulation in MH is not due to an abnormality in calmodulin.     

A high-throughput turbidometric assay for screening inhibitors of Leishmania major protein disulfide isomerase

The use of a high-throughput technique to perform a pilot screen for Leishmania major protein disulfide isomerase (LmPDI) inhibitors identification is reported. In eukaryotic cells, protein disulfide isomerase (PDI) plays a crucial role in protein folding by catalyzing the rearrangement of disulfide bonds in substrate proteins following their synthesis. LmPDI displays similar domain structure organization and functional properties to other PDI family members and is involved in Leishmania virulence. The authors used a method based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol. The screen of a small library of 1920 compounds was performed in a 384-well format and led to the identification of 27 compounds with inhibitory activity against LmPDI. The authors further tested the cytotoxicity of these compounds using Jurkat cells as well as their effect on Leishmania donovani amastigotes using high-content analysis. Results show hexachlorophene and a mixture of theaflavin monogallates inhibit Leishmania multiplication in infected macrophages derived from THP-1 cells, although the inhibitory effect on LmPDI enzymatic activity does not necessarily correlate with the antileishmanial activity.