Expression of the two nested overlapping reading frames of turnip yellow mosaic virus RNA is enhanced by a 5' cap and by 5' and 3' viral sequences

The translation efficiency of an mRNA molecule is typically determined by its 5'- and/or 3'-untranslated regions (UTRs). Previously, we have found that the 3'-UTR of Turnip yellow mosaic virus (TYMV) RNA enhances translation synergistically with a 5' cap. Here, we use a luciferase reporter system in cowpea protoplasts to show that the 5' 217 nucleotides from TYMV genomic RNA enhance expression relative to a vector-derived 17-nucleotide 5'-UTR. Maximum expression was observed from RNAs with a cap and both 5' and 3' TYMV sequences. In paired reporter constructs, the 5' 217 nucleotides harboring the UTR and the first 43 or 41 codons of the two overlapping TYMV open reading frames (ORFs), ORF-69 and ORF-206, respectively, were fused in frame with the luciferase gene. This allowed expression from the initiation codon of each ORF (AUG69 and AUG206) to be monitored separately but from the normal sequence environment. Expression from both AUG codons was heavily dependent on a 5' cap, with a threefold-higher expression occurring from AUG69 than from AUG206 in the presence of the genomic 3'-UTR. Changes that interrupted the cap/3'-UTR synergy (i.e., removal of the cap or TYMV 3'-UTR) resulted in a higher proportion of initiation from AUG206. Mutation of the 3'-UTR to prevent aminoacylation, as well as deletion of 75% of the 5'-UTR, likewise resulted in a lower ratio of expression from AUG69 relative to AUG206. Mutation of each AUG initiation codon increased initiation from the other. Taken together, these results do not fully conform to the expectations of standard leaky ribosomal scanning and leave open the precise mechanism of ribosome commitment to AUG69 and AUG206. However, our observations do not support a recent proposal based on in vitro studies in which the 3'-UTR is proposed to direct cap-independent initiation specifically at AUG206 and not at AUG69 (S. Barends et al., Cell 112:123-129, 2003).     

Provisioning in wild golden lion tamarins (Leontopithecus rosalia): benefits to omnivorous young

Provisioning may act to cushion weaned young from dietary insufficiencyand errors during the period in which they are mastering complexforaging techniques or learning to identify appropriate dietaryitems. That is, young mammals who receive food from others maygain nutritional and/or informational benefits. I conducteda longitudinal study of 13 wild golden lion tamarins 11–56weeks of age in six groups to evaluate hypotheses regardingthe functions of provisioning. All members belonging to thisprimate taxonomic family (the Callitrichidae) are cooperativebreeders and are known to provision their young more frequentlythan do other primate species, except humans. My results, togetherwith experimental findings, suggest that juveniles receive bothnutritional and informational benefits from being provisioned.My juvenile study subjects received animal prey (invertebratesand small vertebrates) from others more frequently than plantresources (fruits and hardened exudates). Apparently difficult-to-handlefruits were more likely to be transferred than readily processedfruits. These results support the nutritional benefits hypothesisbecause the young received items, particularly lipid- and protein-richprey, that they might not otherwise have acquired. That juvenilesfed independently on, and were provisioned with, the same fruitson the same day is counterevidence to the nutritional benefitshypothesis, however. The informational benefits hypothesis wassupported because juveniles received a large variety of foods(including more than 20% of fruit species eaten) and receiveduncommon fruits that were easily acquired. Adults emitted food-offeringcalls to encourage the transfer of prey to juveniles, particularlywhen the prey was whole and alive.     

Scanning cytometry with a LEAP: laser-enabled analysis and processing of live cells in situ.

BACKGROUND: Scanning cytometry now has many of the features (and power) of multiparameter flow cytometry while keeping its own advantages as an imaging technology. Modern instruments combine capabilities of scanning cytometry with the ability to manipulate cells. A new technology, called LEAP (laser-enabled analysis and processing), offers a unique combination of capabilities in cell purification and selective macromolecule delivery (optoinjection). METHODS: LEAP-mediated cell purification and optoinjection effects were assessed in model experiments using adherent and suspension cell types and cell mixtures plated and processed at different densities. Optoinjection effects were visualized by delivering fluorescent dextrans into cells. Results were analyzed using the LEAP instrument's own imaging system as well as by fluorescence and confocal microscopy. RESULTS: Live cell samples (adherent and suspension) could be purified to 90-100% purity with 50-90% yield, causing minimal cell damage depending on the cell type and plating density. Nearly one hundred percent of the targeted cells of all cell types examined could be successfully optoinjected with dextrans of 3-70 kDa, causing no visual damage to the cells. Indirect optoinjection effects were observed on untargeted cells within 5-60 microm to targeted areas under conditions used here. CONCLUSIONS: LEAP provides solutions in cell purification and targeted macromolecule delivery for traditional and challenging applications where other methods fall short.     

Patency Outcomes of Aortic Connectors

OBJECTIVE:: Controlled outcome analysis of mechanical aortic connectors for proximal saphenous vein bypass graft anastomosis is lacking. We report the clinical and angiographic outcome of patients receiving the Symmetry aortic connector (St. Jude Medical, Inc St. Paul, MN, US) within a multicenter, prospective, randomized study. METHODS:: Twenty-five patients at 3 study sites received aortic connectors at the time of coronary artery bypass surgery. Protocol-defined angiographic follow-up was completed in 19 of 25 patients (76%) at time-points up to 14 months postoperatively; 32 connector anastomoses were evaluated in these 19 patients. Beating heart surgery was performed in 17 patients, and 2 were performed with cardiopulmonary bypass. Age was 69.7 ± 8.1 year; all patients were males. RESULTS:: The connector anastomosis patency rate was 15.6% (5/32). There were no deaths during the follow-up period. Four patients (21%) suffered myocardial infarction and 2 additional patients (10.5%) required percutaneous coronary interventions; one of who required 3 percutaneous coronary interventions, the other received one percutaneous coronary intervention. CONCLUSIONS:: In this nonrandomized cohort of patients, occlusion rate with Symmetry connectors was significantly greater than anticipated. Patients who have received these connectors during coronary artery bypass surgery may require closer follow-up and evaluation. While the manufacturer has stopped producing this device, there has been no recall of the product, clinical support remains ongoing, and next generation connectors have now been marketed. Consideration should be given to discontinuation of the clinical use of Symmetry connectors.     

Elevated mutation rates in the germline of Polkappa mutant male mice

Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of DNA polymerase kappa (Polkappa(-/-)) deficient mice. The spontaneous mutation rate in homozygous Polkappa(-/-) males was significantly higher than in isogenic wild-type mice (Polkappa(+/+)), but the ESTR mutation spectrum in Polkappa(-/-) animals did not differ from that in Polkappa(+/+) males. We suggest that compromised translesion synthesis in Polkappa(-/-) mice may result in replication fork pausing which, in turn, may affect ESTR mutation rate.     

The Decline of the Sharp-Snouted Day Frog (Taudactylus acutirostris): The First Documented Case of Extinction by Infection in a Free-Ranging Wildlife Species?

Infectious diseases are increasingly recognized as the cause of mass mortality events, population declines, and the local extirpation of wildlife species. In a number of cases, it has been hypothesized that pathogens have caused species extinctions in wildlife. However, there is only one definitively proven case of extinction by infection, and this was in a remnant captive population of a Polynesian tree snail. In this article, we review the potential involvement of infectious disease in the recent extinction of the sharp-snouted day frog Taudactylus acutirostris. Our review of available evidence suggests that a virulent pathogen of amphibians, Batrachochytrium dendrobatidis, caused a rapid, catastrophic decline of this species, from which it did not recover. We propose that this is the first case of extinction by infection of a free-ranging wildlife species where disease acted as both the proximate and ultimate cause of extinction. This highlights a probable underreporting of infectious disease as a cause of biodiversity loss historically and currently.     

Efficiency of new fungal cellulase systems in boosting enzymatic degradation of barley straw lignocellulose

This study examined the cellulytic effects on steam-pretreated barley straw of cellulose-degrading enzyme systems from the five thermophilic fungi Chaetomium thermophilum, Thielavia terrestris, Thermoascus aurantiacus, Corynascus thermophilus, and Myceliophthora thermophila and from the mesophile Penicillum funiculosum. The catalytic glucose release was compared after treatments with each of the crude enzyme systems when added to a benchmark blend of a commercial cellulase product, Celluclast, derived from Trichoderma reesei and a beta-glucosidase, Novozym 188, from Aspergillus niger. The enzymatic treatments were evaluated in an experimental design template comprising a span of pH (3.5-6.5) and temperature (35-65 degrees C) reaction combinations. The addition to Celluclast + Novozym 188 of low dosages of the crude enzyme systems, corresponding to 10 wt % of the total enzyme protein load, increased the catalytic glucose yields significantly as compared to those obtained with the benchmark Celluclast + Novozyme 188 blend. A comparison of glucose yields obtained on steam-pretreated barley straw and microcrystalline cellulose, Avicel, indicated that the yield improvements were mainly due to the presence of highly active endoglucanase activity/activities in the experimental enzyme preparations. The data demonstrated the feasibility of boosting the widely studied T. reeseicellulase enzyme system with additional enzymatic activity to achieve faster lignocellulose degradation. We conclude that this supplementation strategy appears feasible as a first step in identifying truly promising fungal enzyme sources for fast development of improved, commercially viable, enzyme preparations for lignocellulose degradation.