Validation of the Calypso Surface Beacon Transponder.

Calypso L-shaped Surface Beacon transponder has recently become available for clinical applications. We herein conduct studies to validate the Surface Beacon transponder in terms of stability, reproducibility, orientation sensitivity, cycle rate dependence, and respiratory waveform tracking accuracy. The Surface Beacon was placed on a Quasar respiratory phantom and positioned at the isocenter with its two arms aligned with the lasers. Breathing waveforms were simulated, and the motion of the transponder was tracked. Stability and drift analysis: sinusoidal waveforms (200 cycles) were produced, and the amplitudes of phases 0% (inhale) and 50% (exhale) were recorded at each breathing cycle. The mean and standard deviation (SD) of the amplitudes were calculated. Linear least-squares fitting was performed to access the possible amplitude drift over the breathing cycles. Reproducibility: similar setting to stability and drift analysis, and the phantom generated 100 cycles of the sinusoidal waveform per run. The Calypso system's was re-setup for each run. Recorded amplitude and SD of 0% and 50% phase were compared between runs to assess contribution of Calypso electromagnetic array setup variation. Beacon orientation sensitivity: the Calypso tracks sinusoidal phantom motion with a defined angular offset of the beacon to assess its effect on SD and peak-to-peak amplitude. Rate dependence: sinusoidal motion was generated at cycle rates of 1 Hz, .33 Hz, and .2 Hz. Peak-to-peak displacement and SDs were assessed. Respiratory waveform tracking accuracy: the phantom reproduced recorded breathing cycles (by volunteers and patients) were tracked by the Calypso system. Deviation in tracking position from produced waveform was used to calculate SD throughout entire breathing cycle. Stability and drift analysis: Mean amplitude ± SD of phase 0% or 50% were 20.01 ± 0.04 mm and -19.65 ± 0.08 mm, respectively. No clinically significant drift was detected with drift measured as 5.1 × 10-5 mm/s at phase 0% and -6.0 × 10-5 mm/s at phase 50%. Reproducibility: The SD of the setup was 0.06 mm and 0.02 mm for phases 0% and 50%, respectively. The combined SDs, including both setup and intrarun error of all runs at phases 0% and 50%, were 0.07mm and 0.11 mm, respectively. Beacon orientation: SD ranged from 0.032mm to 0.039 mm at phase 0% and from 0.084 mm to 0.096 mm at phase 50%. The SD was found not to vary linearly with Beacon angle in the range of 0° and 15°. A positive systematic error was observed with amplitude 0.07 mm/degree at phase 0% and 0.05 mm/degree at phase 50%. Rate dependence: SD and displacement amplitudes did not vary significantly between 0.2 Hz and 0.33 Hz. At 1 Hz, both 0% and 50% amplitude measurements shifted up appreciably, by 0.72 mm and 0.78mm, respectively. As compared with the 0.33 Hz data, SD at phase 0% was 1.6 times higher and 5.4 times higher at phase 50%. Respiratory waveform tracking accuracy: SD of 0.233 mm with approximately normal distribution in over 134 min of tracking (201468 data points). The Surface Beacon transponder appears to be stable, accurate, and reproducible. Submillimeter resolution is achieved throughout breathing and sinusoidal waveforms.

Growth rate and cell size modulate the synthesis of, and requirement for, G1-phase cyclins at start.

In Saccharomyces cerevisiae, commitment to cell cycle progression occurs at Start. Progression past Start requires cell growth and protein synthesis, a minimum cell size, and G(1)-phase cyclins. We examined the relationships among these factors. Rapidly growing cells expressed, and required, dramatically more Cln protein than did slowly growing cells. To clarify the role of cell size, we expressed defined amounts of CLN mRNA in cells of different sizes. When Cln was expressed at nearly physiological levels, a critical threshold of Cln expression was required for cell cycle progression, and this critical threshold varied with both cell size and growth rate: as cells grew larger, they needed less CLN mRNA, but as cells grew faster, they needed more Cln protein. At least in part, large cells had a reduced requirement for CLN mRNA because large cells generated more Cln protein per unit of mRNA than did small cells. When Cln was overexpressed, it was capable of promoting Start rapidly, regardless of cell size or growth rate. In summary, the amount of Cln required for Start depends dramatically on both cell size and growth rate. Large cells generate more Cln1 or Cln2 protein for a given amount of CLN mRNA, suggesting the existence of a novel posttranscriptional size control mechanism.

RNA interference and heterochromatin assembly.

In most eukaryotes, histone and DNA modifications are responsible for the silencing of genes integrated in heterochromatic sequences, as well as the silencing of pericentromeric repeats and transposable elements themselves. But the mechanisms that guide these modifications to heterochromatin during the cell cycle have been elusive. RNA interference takes advantage of heterochromatic transcription to process small RNAs and recruit enzymes required for both histone and DNA modifications, and is one such mechanism that has been identified. The processes are best understood in fission yeast and plants, but recent work in mammalian cells, especially in the germline, suggests these mechanisms may be highly conserved.

DNA zip codes control an ancient mechanism for gene targeting to the nuclear periphery.

Many genes in Saccharomyces cerevisiae are recruited to the nuclear periphery after transcriptional activation. We have identified two gene recruitment sequences (GRS I and II) from the promoter of the INO1 gene that target the gene to the nuclear periphery. These GRSs function as DNA zip codes and are sufficient to target a nucleoplasmic locus to the nuclear periphery. Targeting requires components of the nuclear pore complex (NPC) and a GRS is sufficient to confer a physical interaction with the NPC. GRS I elements are enriched in promoters of genes that interact with the NPC, and genes that are induced by protein folding stress. Full transcriptional activation of INO1 and another GRS-containing gene requires GRS-mediated targeting of the promoter to the nuclear periphery. Finally, GRS I also functions as a DNA zip code in Schizosaccharomyces pombe, suggesting that this mechanism of targeting to the nuclear periphery has been conserved over approximately one billion years of evolution.

RNA interference is required for normal centromere function in fission yeast.

In plants, animals and fungi, active centromeres are associated with arrays of repetitive DNA sequences. The outer repeats at fission yeast (Schizosaccharomyces pombe) centromeres are heterochromatic and are required for the assembly of an active centromere. Components of the RNA interference (RNAi) machinery process transcripts derived from these repeats and mediate the formation of silent chromatin. A subfragment of the repeat (dg) is known to induce silencing of marker genes at euchromatic sites and is required for centromere formation. We show that the RNAi components, Argonaute (Ago1), Dicer (Dcr1) and RNA-dependent RNA polymerase (Rdp1), are required to maintain silencing, lysine 9 methylation of histone H3 and association of Swi6 via this dg ectopic silencer. Deletion of Ago1, Dcr1 or Rdp1 disrupts chromosome segregation leading to a high incidence of lagging chromosomes on late anaphase spindles and sensitivity to a microtubule poison. Analysis of dg transcription indicates that csp mutants, previously shown to abrogate centromere silencing and chromosome segregation, are also defective in the regulation of non-coding centromeric RNAs. In addition, histone H3 lysine 9 methylation at, and recruitment of Swi6 and cohesin to, centromeric repeats is disrupted in these mutants. Thus the formation of silent chromatin on dg repeats and the development of a fully functional centromere is dependent on RNAi.