The presence of low-copy-number regulators and switch-like signal propagation in regulatory networks are expected to increase noise in cellular processes. We developed a noise amplifier that detects fluctuations in the level of low-abundance mRNAs in yeast. The observed fluctuations are not due to the low number of molecules expressed from a gene per se but originate in the random, rare events of gene activation. The frequency of these events and the correlation between stochastic expressions of genes in a single cell depend on the positioning of the genes along the chromosomes. Transcriptional regulators produced by such random expression propagate noise to their target genes.

During Mycobacterium tuberculosis infection, a population of bacteria likely becomes refractory to antibiotic killing in the absence of genotypic resistance, making treatment challenging. We describe an in vitro model capable of yielding a phenotypically antibiotic-tolerant subpopulation of cells, often called persisters, within populations of Mycobacterium smegmatis and M. tuberculosis . We find that persisters are distinct from the larger antibiotic-susceptible population, as a small drop in dissolved oxygen (DO) saturation (20%) allows for their survival in the face of bactericidal antibiotics. In contrast, if high levels of DO are maintained, all cells succumb, sterilizing the culture. With increasing evidence that bactericidal antibiotics induce cell death through the production of reactive oxygen species (ROS), we hypothesized that the drop in DO decreases the concentration of ROS, thereby facilitating persister survival, and maintenance of high DO yields sufficient ROS to kill persisters. Consistent with this hypothesis, the hydroxyl-radical scavenger thiourea, when added to M. smegmatis cultures maintained at high DO levels, rescues the persister population. Conversely, the antibiotic clofazimine, which increases ROS via an NADH-dependent redox cycling pathway, successfully eradicates the persister population. Recent work suggests that environmentally induced antibiotic tolerance of bulk populations may result from enhanced antioxidant capabilities. We now show that the small persister subpopulation within a larger antibiotic-susceptible population also shows differential susceptibility to antibiotic-induced hydroxyl radicals. Furthermore, we show that stimulating ROS production can eradicate persisters, thus providing a potential strategy to managing persistent infections.

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The partitioning and subsequent inheritance of cellular factors like proteins and RNAs is a ubiquitous feature of cell division. However, direct quantitative measures of how such nongenetic inheritance affects subsequent changes in gene expression have been lacking. We tracked families of the yeast Saccharomyces cerevisiae as they switch between two semi-stable epigenetic states. We found that long after two cells have divided, they continued to switch in a synchronized manner, whereas individual cells have exponentially distributed switching times. By comparing these results to a Poisson process, we show that the time evolution of an epigenetic state depends initially on inherited factors, with stochastic processes requiring several generations to decorrelate closely related cells. Finally, a simple stochastic model demonstrates that a single fluctuating regulatory protein that is synthesized in large bursts can explain the bulk of our results.

With rising rates of drug-resistant infections, there is a need for diagnostic methods that rapidly can detect the presence of pathogens and reveal their susceptibility to antibiotics. Here we propose an approach to diagnosing the presence and drug-susceptibility of infectious diseases based on direct detection of RNA from clinical samples. We demonstrate that species-specific RNA signatures can be used to identify a broad spectrum of infectious agents, including bacteria, viruses, yeast, and parasites. Moreover, we show that the behavior of a small set of bacterial transcripts after a brief antibiotic pulse can rapidly differentiate drug-susceptible and -resistant organisms and that these measurements can be made directly from clinical materials. Thus, transcriptional signatures could form the basis of a uniform diagnostic platform applicable across a broad range of infectious agents.

Commercial EUV lithographic systems require multilayers with higher reflectance and better stability then that published to date. Interface-engineered MoISi multilayers with 70% reflectance at 13.5 nm wavelength (peak width of 0.545 nm) and 71% at 12.7 nm wavelength (peak width of 0.49 nm) were developed. These results were achieved with 50 bilayers. These new multilayers consist of Mo and Si layers separated by thin boron carbide layers. Depositing boron carbide on interfaces leads to reduction in silicide formation on the Mo-on-Si interfaces. Bilayer contraction is reduced by 30% implying that there is less intermixing of Mo and Si to form silicide. As a result the Mo-on-Si interfaces are sharper in interface-engineered multilayers than in standard Mo/Si multilayers. The optimum boron carbide thicknesses have been determined and appear to be different for Mo-on-Si and Si-on-Mo interfaces. The best results were obtained with 0.4 nm thick boron carbide layer on the Mo-on-Si interface and 0.25 nm thick boron carbide layer on the Si-on-Mo interface. Increase in reflectance is consistent with multilayers with sharper and smoother interfaces.A significant improvement in oxidation resistance of EUV multilayers has been achieved with ruthenium terminated MoISi multilayers. The best capping layer design consists of a Ru layer separated from the last Si layer by a boron carbide layer. This design achieves high reflectance and the best oxidation resistance in a water vapor (i.e. oxidation) environment. Electron beam exposures of 4.5 hours in the presence of 5~1 0 -~ torr water vapor partial pressure show no measurable reflectance loss and no increase in the oxide thickness of Ru terminated multilayers. Longer exposures in different environments are necessary to test lifetime stability of many years.. I

In order to satisfy the metrology requirements of multilayer coatings for EUVL optics and masks, improvements have been made to the reflectometry beamline at the Advanced Light Source. The precision in determining multilayer peak reflectance and wavelength has been improved by reducing the measurement noise. The peak reflectance of a typical Mo/Si multilayer can now be measured with a precision of 0.08% rms (relative) and the centroid wavelength with a precision of 0.007% rms. It has now been possible to determine the contribution of scattered light to the spectral purity.Under the typical measurement conditions the scattered light accounts for about 1.3% of the incident beam. With an appropriate slit it is possible to reduce the scattered light to 0.25%. By correcting for the remaining scattered light, it is estimated that a reflectance accuracy of 0.1% (absolute) is obtained for a typical Mo/Si multilayer.

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