Search for Limiting Factors in the RNAi Pathway in Silkmoth Tissues and the Bm5 Cell Line: The RNA-Binding Proteins R2D2 and Translin

RNA interference (RNAi), an RNA-dependent gene silencing process that is initiated by double-stranded RNA (dsRNA) molecules, has been applied with variable success in lepidopteran insects, in contrast to the high efficiency achieved in the coleopteran Tribolium castaneum. To gain insight into the factors that determine the efficiency of RNAi, a survey was carried out to check the expression of factors that constitute the machinery of the small interfering RNA (siRNA) and microRNA (miRNA) pathways in different tissues and stages of the silkmoth, Bombyx mori. It was found that the dsRNA-binding protein R2D2, an essential component in the siRNA pathway in Drosophila, was expressed at minimal levels in silkmoth tissues. The silkmoth-derived Bm5 cell line was also deficient in expression of mRNA encoding full-length BmTranslin, an RNA-binding factor that has been shown to stimulate the efficiency of RNAi. However, despite the lack of expression of the RNA-binding proteins, silencing of a luciferase reporter gene was observed by co-transfection of luc dsRNA using a lipophilic reagent. In contrast, gene silencing was not detected when the cells were soaked in culture medium supplemented with dsRNA. The introduction of an expression construct for Tribolium R2D2 (TcR2D2) did not influence the potency of luc dsRNA to silence the luciferase reporter. Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells. Our results offer a first evaluation of the expression of the RNAi machinery in silkmoth tissues and Bm5 cells and provide evidence for a functional RNAi response to intracellular dsRNA in the absence of R2D2 and Translin. The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed

Molecular imaging of rheumatoid arthritis by radiolabelled monoclonal antibodies: new imaging strategies to guide molecular therapies

The closing of the last century opened a wide variety of approaches for inflammation imaging and treatment of patients with rheumatoid arthritis (RA). The introduction of biological therapies for the management of RA started a revolution in the therapeutic armamentarium with the development of several novel monoclonal antibodies (mAbs), which can be murine, chimeric, humanised and fully human antibodies. Monoclonal antibodies specifically bind to their target, which could be adhesion molecules, activation markers, antigens or receptors, to interfere with specific inflammation pathways at the molecular level, leading to immune-modulation of the underlying pathogenic process. These new generation of mAbs can also be radiolabelled by using direct or indirect method, with a variety of nuclides, depending upon the specific diagnostic application. For studying rheumatoid arthritis patients, several monoclonal antibodies and their fragments, including anti-TNF-α, anti-CD20, anti-CD3, anti-CD4 and anti-E-selectin antibody, have been radiolabelled mainly with 99mTc or 111In. Scintigraphy with these radiolabelled antibodies may offer an exciting possibility for the study of RA patients and holds two types of information: (1) it allows better staging of the disease and diagnosis of the state of activity by early detection of inflamed joints that might be difficult to assess; (2) it might provide a possibility to perform ‘evidence-based biological therapy’ of arthritis with a view to assessing whether an antibody will localise in an inflamed joint before using the same unlabelled antibody therapeutically. This might prove particularly important for the selection of patients to be treated since biological therapies can be associated with severe side-effects and are considerably expensive. This article reviews the use of radiolabelled mAbs in the study of RA with particular emphasis on the use of different radiolabelled monoclonal antibodies for therapy decision-making and follow-up

A neighborhood statistics model for predicting stream pathogen indicator levels

Because elevated levels of water-borne Escherichia coli in streams are a leading cause of water quality impairments in the U.S., water-quality managers need tools for predicting aqueous E. coli levels. Presently, E. coli levels may be predicted using complex mechanistic models that have a high degree of unchecked uncertainty or simpler statistical models. To assess spatio-temporal patterns of instream E. coli levels, herein we measured E. coli, a pathogen indicator, at 16 sites (at four different times) within the Squaw Creek watershed, Iowa, and subsequently, the Markov Random Field model was exploited to develop a neighborhood statistics model for predicting instream E. coli levels. Two observed covariates, local water temperature (degrees Celsius) and mean cross-sectional depth (meters), were used as inputs to the model. Predictions of E. coli levels in the water column were compared with independent observational data collected from 16 in-stream locations. The results revealed that spatio-temporal averages of predicted and observed E. coli levels were extremely close. Approximately 66 % of individual predicted E. coli concentrations were within a factor of 2 of the observed values. In only one event, the difference between prediction and observation was beyond one order of magnitude. The mean of all predicted values at 16 locations was approximately 1 % higher than the mean of the observed values. The approach presented here will be useful while assessing instream contaminations such as pathogen/pathogen indicator levels at the watershed scale

Incidence and Tracking of Escherichia coli O157:H7 in a Major Produce Production Region in California

Fresh vegetables have become associated with outbreaks caused by Escherichia coli O157:H7 (EcO157). Between 1995–2006, 22 produce outbreaks were documented in the United States, with nearly half traced to lettuce or spinach grown in California. Outbreaks between 2002 and 2006 induced investigations of possible sources of pre-harvest contamination on implicated farms in the Salinas and San Juan valleys of California, and a survey of the Salinas watershed. EcO157 was isolated at least once from 15 of 22 different watershed sites over a 19 month period. The incidence of EcO157 increased significantly when heavy rain caused an increased flow rate in the rivers. Approximately 1000 EcO157 isolates obtained from cultures of>100 individual samples were typed using Multi-Locus Variable-number-tandem-repeat Analysis (MLVA) to assist in identifying potential fate and transport of EcO157 in this region. A subset of these environmental isolates were typed by Pulse Field Gel Electrophoresis (PFGE) in order to make comparisons with human clinical isolates associated with outbreak and sporadic illness. Recurrence of identical and closely related EcO157 strains from specific locations in the Salinas and San Juan valleys suggests that transport of the pathogen is usually restricted. In a preliminary study, EcO157 was detected in water at multiple locations in a low-flow creek only within 135 meters of a point source. However, possible transport up to 32 km was detected during periods of higher water flow associated with flooding. During the 2006 baby spinach outbreak investigation, transport was also detected where water was unlikely to be involved. These results indicate that contamination of the environment is a dynamic process involving multiple sources and methods of transport. Intensive studies of the sources, incidence, fate and transport of EcO157 near produce production are required to determine the mechanisms of pre-harvest contamination and potential risks for human illness

MAGIC and Fermi-LAT gamma-ray results on unassociated HAWC sources

The HAWC Collaboration released the 2HWC catalogue of TeV sources, in which 19 show no association with any known high-energy (HE; E greater than or similar to 10 GeV) or very-high-energy (VHE; E greater than or similar to 300 GeV) sources. This catalogue motivated follow-up studies by both the Major Atmospheric Gamma-ray Imaging Cherenkov (MAGIC) and Fermi-LAT (Large Area Telescope) observatories with the aim of investigating gamma-ray emission over a broad energy band. In this paper, we report the results from the first joint work between High Altitude Water Cherenkov (HAWC), MAGIC, and Fermi-LAT on three unassociated HAWC sources: 2HWC J2006+341, 2HWC J1907+084*, and 2HWC J1852+013*. Although no significant detection was found in the HE and VHE regimes, this investigation shows that a minimum 1 degrees extension (at 95 per cent confidence level) and harder spectrum in the GeV than the one extrapolated from HAWC results are required in the case of 2HWC J1852+013*, whilst a simply minimum extension of 0.16 degrees (at 95 per cent confidence level) can already explain the scenario proposed by HAWC for the remaining sources. Moreover, the hypothesis that these sources are pulsar wind nebulae is also investigated in detail