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Stress granules (SGs) are ribonucleoprotein (RNP) assemblies that form in eukaryotic cells as a result of limited translation in response to stress. SGs form during viral infection and are thought to promote the antiviral response because many viruses encode inhibitors of SG assembly. However, the antiviral endoribonuclease RNase L also alters SG formation, whereby only small punctate SG-like bodies that we term RNase L-dependent bodies (RLBs) form during RNase L activation. How RLBs relate to SGs and their mode of biogenesis is unknown. Herein, using immunofluorescence, live-cell imaging, and MS-based analyses, we demonstrate that RLBs represent a unique RNP granule with a protein and RNA composition distinct from that of SGs in response to dsRNA lipofection in human cells. We found that RLBs are also generated independently of SGs and the canonical dsRNA-induced SG biogenesis pathway, because RLBs did not require protein kinase R, phosphorylation of eukaryotic translation initiation factor 2 subunit 1 (eIF2α), the SG assembly G3BP paralogs, or release of mRNAs from ribosomes via translation elongation. Unlike the transient interactions between SGs and P-bodies, RLBs and P-bodies extensively and stably interacted. However, despite both RLBs and P-bodies exhibiting liquid-like properties, they remained distinct condensates. Taken together, these observations reveal that RNase L promotes the formation of a unique RNP complex that may have roles during the RNase L-mediated antiviral response.

Eukaryotic cells have evolved multiple quality control mechanisms that recognize and eliminate defective mRNA during the process of translation. One mechanism, referred to as No-go decay (NGD), targets mRNAs with elongation stalls for degradation initiated by endonucleolytic cleavage in the vicinity of the stalled ribosome. NGD is promoted by the evolutionarily conserved Dom34 and Hbs1 proteins, which are related to the translation termination factors eRF1 and eRF3, respectively. NGD is likely to occur by Dom34/Hbs1 interacting with the A site in the ribosome leading to release of the peptide or peptidyl-tRNA. The process of NGD and/or the function of Dom34/Hbs1 appear to be important in several different biological contexts.

Ski8p is a WD-repeat protein with an essential role for the Ski complex assembly in an exosome-dependent 3'-to-5' mRNA decay. In addition, Ski8p is involved in meiotic recombination by interacting with Spo11p protein. We have determined the crystal structure of Ski8p from Saccharomyces cerevisiae at 2.2 A resolution. The structure reveals that Ski8p folds into a seven-bladed beta propeller. Mapping sequence conservation and hydrophobicities of amino acids on the molecular surface of Ski8p reveals a prominent site on the top surface of the beta propeller, which is most likely involved in mediating interactions of Ski8p with Ski3p and Spo11p. Mutagenesis combined with yeast two-hybrid and GST pull-down assays identified the top surface of the beta propeller as being required for Ski8p binding to Ski3p and Spo11p. The functional implications for Ski8p function in both mRNA decay and meiotic recombination are discussed.

Using an F 1 screen, 352 feral individuals of the sugarcane borer, Diatraea saccharalis (F.) (Lepidoptera: Crambidae), were examined for the presence of Bacillus thuringiensis (Bt)-resistance alleles. These insects represented four geographical populations collected in central and northeastern Louisiana, USA, and one field population from the Gulf Coast area of Texas, USA, during 2006. The F 1 screen used various crosses between field-collected insects and a laboratory strain of CrylAb-resistant D. saccharalis, including both reciprocal crosses and group mating. F 1 neonates of the crosses were screened for Bt resistance on Bt maize leaf tissue. One field-collected individual of D. saccharalis was shown to have a Bt-resistance allele. Based on Bayesian analysis procedures, the Bt-resistance allele frequency in the five populations of D. saccharalis was 0.0028 with a 95% confidence interval of 0.0003-0.0079. The successful identification of a resistance allele in a field collection of insects suggests that the F 1 screening technique could be an effective tool for detecting and monitoring rare Bt-resistance alleles in field populations of D. saccharalis.

We conducted two experiments to determine the most appropriate method for use in a mark–recapture study of movements of the mummichog Fundulus heteroclitus in the southern Gulf of St. Lawrence. In the first experiment, 315 mummichogs were marked with fin clips, fingerling tags, acrylic paint, or visible implant elastomer (VIE) and were held for 144 d. Percent tag retention (98.5%) and survival (100%) were highest for the VIE group, and no negative effect was observed on length growth. This led to a more detailed experiment (167 d) to assess potential negative effects of VIE on the growth and survival of 144 mummichogs marked on various body locations; we also assessed mark retention and readability in this experiment. No negative effects on growth were detected; mean daily length increases ranged from 0.04 to 0.12 mm, and specific growth rates for all groups ranged from 0.129% to 0.470%. Mean survival of all groups was 90.8% (range = 66.7–100%) and was higher in marked groups than controls. All m...

A major mechanism of eukaryotic mRNA degradation initiates with deadenylation followed by decapping and 5' to 3' degradation. We demonstrate that the yeast EDC1 mRNA, which encodes a protein that enhances decapping, has unique properties and is both protected from deadenylation and undergoes deadenylation-independent decapping. The 3' UTR of the EDC1 mRNA is sufficient for both protection from deadenylation and deadenylation-independent decapping and an extended poly(U) tract within the 3' UTR is required. These observations highlight the diverse forms of decapping regulation and identify a feedback loop that can compensate for decreases in activity of the decapping enzyme. Surprisingly, the decapping of the EDC1 mRNA is slowed by the loss of Not2p, Not4p, and Not5p, which interact with the Ccr4p/Pop2p deadenylase complex. This indicates that the Not proteins can affect decapping, which suggests a possible link between the mRNA deadenylation and decapping machinery.

The major pathway of mRNA degradation in yeast occurs through deadenylation, decapping and subsequent 5′ to 3′ exonucleolytic decay of the transcript body. To identify proteins that control the activity of the decapping enzyme, which is encoded by the DCP1 gene, we isolated a high‐copy suppressor of the temperature‐sensitive dcp1‐2 allele, termed DCP2. Overexpression of Dcp2p partially suppressed the dcp1‐2 decapping defect. Moreover, the Dcp2 protein was required for the decapping of both normal mRNAs and aberrant transcripts that are degraded by the mRNA surveillance pathway. The Dcp2 protein contains a MutT motif, which is found in a class of pyrophosphatases. Mutational analyses indicated that the region of Dcp2p containing the MutT motif is necessary and sufficient for Dcp2p9s function in mRNA decapping. The Dcp2p also coimmunoprecipitates with the DCP1 decapping enzyme and is required for the production of enzymatically active decapping enzyme. These results suggest that direct or indirect interaction of Dcp1p with Dcp2p is required for the production of active decapping enzyme, perhaps in a process requiring the hydrolysis of a pyrophosphate bond.

Measuring the outcomes of social work interventions with children raises complex issues. The instability of intermediate outcomes emphasises the need to undertake regular and carefully timed assessments. Differences in approach and perspective can obscure relationships between evaluations of organisational performance and assessments of outcome for individual children; user perspectives introduce another point of view. Assessing outcomes of family support services raises questions of accountability. Aggregate information about children looked after, collected through the Looking After Children project, should help to establish the chances of certain outcomes being realised, although the influence of some factors may not be easy to explain.

Medical Physicists are frequently involved in shipping radioactive materials or supervising those who do. Current U.S. Department of Transportation Hazardous Material Regulations, 49 CFR Parts 171 – 185, require hazmat employees to have documented training specified in 49 CFR 172 Subpart H. A hazmat employee is defined as an individual who: (1) loads, unloads or handles hazardous material; (2) manufactures, tests, reconditions, repairs, modifies, marks or otherwise represents containers, drums or packagings as qualified for use in the transportation of hazardous materials; (3) prepares hazardous materials for transportation; (4) is responsible for safety of transporting hazardous materials; or (5) operates a vehicle used to transport hazardous materials. Recurrent training is required at least once every three years. (The IATA two year training interval is not applicable and is generally misunderstood.) FAA has escalated inspection and enforcement. Facilities who ship radiopharmaceuticals to other laboratories, return radiopharmaceuticals or radioactive sources to suppliers, or otherwise ship radioactive materials have been cited for failure to provide and document the required training. The interrelationship of transportation regulations, 49 CFR, IATA, ICAO and other transportation regulations, which are frequently misunderstood will be explained. The course will cover typical shipments by air and highway which are encountered in a medical institution. Items such as fissile materials, highway route controlled quantities, rail shipments, vessel shipments and such will be omitted; although specific questions may be addressed. A major objective of the course is to present the process of shipping radioactive material in a sequential and logical fashion. How radioactive materials for transportation purposes are defined by activity concentrations for exempt materials and activity limits for exempt consignments will be explained. Radioactive material shipments of excepted packages and Type A packages will be emphasized. The program is designed to meet the general awareness and function specific DOT training requirements for shippers of medical radioactive materials. Safety training and security awareness training is generally satisfied by the training required under the institution's radioactive material license. For shippers of radioactive Yellow III labeled packages an in‐depth written security plan and training are no longer required as of April 8, 2010. In general almost all shippers of medical radioactive material are now not required to have an in‐depth security plan. Contents of security awareness training and in‐depth security plans will be briefly outlined. It is the hazmat employer's responsibility to ensure that each hazmat employee is properly trained. No third party can fulfill that requirement. It is the hazmat employer's responsibility to determine the degree to which this course meets the employer's requirements, including contents of the course and the examination. Participants will gain sufficient knowledge to prepare hazmat training programs for others in their institutions. A handout will be posted which should be printed out and brought to the course for reference during the presentation. The handout will also satisfy part of the training documentation required by DOT. A feature handout section is a composite table which provides A1, A2, RQ, Exempt Concentration, and Exempt Consignment values in a single table in both Becquerel and Curie units. Course attendance will be certified through the AAPM CEU documentation system. Learning Objectives: 1. Understand the regulatory requirements for shipping radioactive materials. 2. Understand the regulatory requirements for training of hazmat employees. 3. Comprehend how to classify, package, mark, label, document, placard, and transport radioactive materials.