Cell cycle regulation of the E2F transcription factor involves an interaction with cyclin A.

We have examined E2F binding activity in extracts of synchronized NIH 3T3 cells. During the G0 to G1 transition, there is a marked increase in the level of active E2F. Subsequently, there are changes in the nature of E2F-containing complexes. A G1-specific complex increases in abundance, disappears, and is then replaced by another complex as S phase begins. Analysis of extracts of thymidine-blocked cells confirms that the complexes are cell cycle regulated. We also show that the cyclin A protein is a component of the S phase complex. Each complex can be dissociated by the adenovirus E1A 12S product, releasing free E2F. The release of E2F from the cyclin A complex coincides with the stimulation of an E2F-dependent promoter. We suggest that these interactions control the activity of E2F and that disruption of the complexes by E1A contributes to a loss of cellular proliferation control.     

Codon usage bias and the evolution of influenza A viruses. Codon Usage Biases of Influenza Virus

Background  

The influenza A virus is an important infectious cause of morbidity and mortality in humans and was responsible for 3 pandemics in the 20^th century. As the replication of the influenza virus is based on its host's machinery, codon usage of its viral genes might be subject to host selection pressures, especially after interspecies transmission. A better understanding of viral evolution and host adaptive responses might help control this disease.     

Silicates,Silicate Weathering,and Microbial Ecology

Mineralogy, microbial ecology, and mineral weathering in the subsurface are an intimately linked biogeochemical system. Although bacteria have been implicated indirectly in the accelerated weathering of minerals, it is not clear if this interaction is simply the coincidental result of microbial metabolism, or if it represents a specific strategy offering the colonizing bacteria a competitive ecological advantage. Our studies provide evidence that silicate weathering by bacteria is sometimes driven by the nutrient requirements of the microbial consortium, and therefore depends on the trace nutrient content of each aquifer mineral. This occurrence was observed in reducing groundwaters where carbon is abundant but phosphate is scarce; here, even resistant feldspars are weathered rapidly. This suggests that the progression of mineral weathering may be influenced by a mineral's nutritional potential, with microorganisms destroying only beneficial minerals. The rock record, therefore, may contain a remnant mineralogy that reflects early microbial destruction of biologically valuable minerals, leaving a residuum of "useless" minerals, where "value" depends on the organism, its metabolic needs, and the diagenetic environment. Conversely, the subsurface distribution of microorganisms may, in part, be controlled by the mineralogy and by the ability of an organism to take advantage of mineral-bound nutrients.     

Locating the broad-spectrum wheat leaf rust resistance gene Lr52 (LrW) to chromosome 5B by a new cytogenetic method

This study was conducted to genetically map a potentially new wheat leaf rust resistance gene (LrW) using a novel genetic method and to test its effectiveness against current races of leaf rust (Puccinia triticina Eriks.) in Canada. Undoubled haploids of a near-isogenic line of Thatcher carrying the resistance gene (RL6107) were pollinated with a contrasting susceptible cultivar to generate an array of hybrids with random deficiencies arising from irregular meiosis of the haploid. Genetic analysis of the deficiencies in such populations can be used to locate qualitative traits by which the two parents differ through a process that we have called haploid deficiency mapping. In the present case, 5/417 hybrids were both susceptible to leaf rust (i.e. lacked the resistance gene) and also lacked several polymorphic microsatellite alleles from RL6107 that are specific to chromosome 5B. This correlated failed transmission of the resistance gene and deficiency for chromosome 5B. Analysis of an F₂ population showed that the factor conditioning resistance was located on the short arm of 5B, 16.5 cM distal to the locus of the microsatellite Xgwm443. Since no other leaf rust resistance genes have been mapped to this region, LrW was re-designated Lr52. RL6107 was tested with 29 isolates of P. triticina, encompassing a diversity of virulence found in North America, with none showing virulence. The effectiveness and novelty of Lr52 make it a promising source of resistance for North American wheat cultivars.     

Autofluorescence imaging identifies tumor cell‐cycle status on a single‐cell level

The goal of this study is to validate fluorescence intensity and lifetime imaging of metabolic co‐enzymes NAD(P)H and FAD (optical metabolic imaging, or OMI) as a method to quantify cell‐cycle status of tumor cells. Heterogeneity in tumor cell‐cycle status (e. g. proliferation, quiescence, apoptosis) increases drug resistance and tumor recurrence. Cell‐cycle status is closely linked to cellular metabolism. Thus, this study applies cell‐level metabolic imaging to distinguish proliferating, quiescent, and apoptotic populations. Two‐photon microscopy and time‐correlated single photon counting are used to measure optical redox ratio (NAD(P)H fluorescence intensity divided by FAD intensity), NAD(P)H and FAD fluorescence lifetime parameters. Redox ratio, NAD(P)H and FAD lifetime parameters alone exhibit significant differences (p<0.05) between population means. To improve separation between populations, linear combination models derived from partial least squares ‐ discriminant analysis (PLS‐DA) are used to exploit all measurements together. Leave‐one‐out cross validation of the model yielded high classification accuracies (92.4 and 90.1 % for two and three populations, respectively). OMI and PLS‐DA also identifies each sub‐population within heterogeneous samples. These results establish single‐cell analysis with OMI and PLS‐DA as a label‐free method to distinguish cell‐cycle status within intact samples. This approach could be used to incorporate cell‐level tumor heterogeneity in cancer drug development.

     

Investigation of Design Space for Freeze-Drying: Use of Modeling for Primary Drying Segment of a Freeze-Drying Cycle

In this work, we explore the idea of using mathematical models to build design space for the primary drying portion of freeze-drying process. We start by defining design space for freeze-drying, followed by defining critical quality attributes and critical process parameters. Then using mathematical model, we build an insilico design space. Input parameters to the model (heat transfer coefficient and mass transfer resistance) were obtained from separate experimental runs. Two lyophilization runs are conducted to verify the model predictions. This confirmation of the model predictions with experimental results added to the confidence in the insilico design space. This simple step-by-step approach allowed us to minimize the number of experimental runs (preliminary runs to calculate heat transfer coefficient and mass transfer resistance plus two additional experimental runs to verify model predictions) required to define the design space. The established design space can then be used to understand the influence of critical process parameters on the critical quality attributes for all future cycles.