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Why You Should Never Leave Your Garage Without Duct Tape in Your Car
Astronauts won't leave earth without duct tape (it saved three astronauts aboard Apollo 13). And here's why you shouldn't leave your garage without it.
1. Emergency Fix
Remember "Houston, we've had a problem"? You probably won't need to make an emergency CO2 scrubber in space, but duct tape is great for automotive emergencies. Is your side mirror dangling after going through the car wash? Duct tape it back in place. Did you get rear-ended, and your bumper is dragging? Duct tape is a great "holder-upper." Smashed taillight? Duct tape can keep the pieces together.
2. Lint Brush
Have you ever been about to get out of your car and then noticed you had pet hair all over your black pants? Duct tape substitutes nicely for a lint roller.
3. First Aid
Duct tape can be used to make bandages, slings, and splints that will suffice until you can get proper medical care. If you use it as a bandage, you might cover the wound first with a piece of a paper towel or tissue and then apply the duct tape.
4. Radiator Hose Repair
A hole in your radiator hose will result in coolant gushing from the cooling system, which will cause the engine to quickly overheat. Once the system has completely cooled off, duct tape can be a perfect temporary patch so that you aren't stranded on the side of the road.
5. MacGyver Options
You can use duct tape to cover a blister, waterproof shoes, identify your luggage, or to make a cup, a cup holder, or even a stretcher!
Some people claim that if you can't fix the problem with duct tape, you're just not using enough. Bottom line: don't leave home without duct tape in your vehicle.


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VOLLEYBALL: Licking volleyball overpowers St. James
|Photo by Shari Harris -- Kamryn Barnes goes up for a spike in Monday night's win.
By Shari Harris
|Photo by Shari Harris -- Maggie Cline serves
in the JV match against St. James.
Junior varsity also pulled out a win with scores of 25-14 and 29-27 against St. James Sept. 17.

subset: commoncrawl


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The idea for 2020 Women on Boards was brought to life in 2010 in Boston. Co-founders Stephanie Sonnabend and Malli Gero, were neighbors and friends who shared similar frustration with the slow progress being made by corporations to increase the numbers of women directors on boards. As former CEO of an international hospitality company, Stephanie Sonnabend has served as a director on two public boards and is a member of women's leadership organizations. Malli Gero, a public relations executive, had worked on women's leadership issues for many years before launching 2020WOB, and has served on a number of nonprofit boards.
The founders wanted to create a grassroots campaign to raise awareness of the issue among young people, middle managers, senior executives, and consumers—people who probably had not previously thought about its relevance to their lives and jobs. They wanted to enlist them as champions through the use of social and traditional media. From that concept, the campaign expanded to involve corporations throughout the country. They tracked the lack of women on boards through in-depth research, and spawned major public events in key cities to educate women at all stages of their careers about getting on boards.
They believe their goal would be easily achievable in ten years: to raise the percentage of women on U.S. corporate boards to 20% or greater by the year 2020. The campaign expanded nationally in 2011.
For the first six years, the campaign tracked the Fortune 1000 corporations and saw the percentage of women on boards increase from 14.6% in 2011 to over 20% in 2017. Having reached the goal of 20% in the Fortune 1000 the campaign transitioned to the Russell 3000 companies. In 2017 the percentage of women on the Russell 3000 was 16% and one-fourth of the companies had no women on their boards. By 2019 the Russell 3000 list exceeded 20% and only 11% of the companies have no women.
Continuing to grow in size and influence over the years, 2020WOB built an army of active volunteers and supporters who continue to propel the campaign forward. When the milestone year 2020 is reached, there will be a new name, a new goal, and renewed focus to add more women on corporate boards.
We thank Stephanie and Malli for their energy, dedication and unstoppable commitment to the long-term increase of women directors on corporate boards and the success of 2020WOB!
2020 Women on Boards is a 501c3 organization. All donations to 2020 Women on Boards are tax deductible to the extent allowed by law.


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Hike to Stillhouse Falls
1 miles 236 ft gain - Out-and-Back Trail
Added by Katie Yarborough
This is a beautiful waterfall, only 1 hour south from Nashville! Easier hike, dog-friendly, and a great escape from the city!
This waterfall is a fun short hike that can be done quick, or a nice place to hangout for the day in your hammock to read a book!
From Nashville, take I-65 south and take the Saturn Parkway toward Columbia. Turn right on Hwy 43 and proceed about 21 miles. The natural area parking area is on the right once you have passed over the Judge Workman Bridge. It is about three miles northeast of Summertown on Hwy 43 on the left just before the bridge. Putting in "Stillhouse Falls" in your gps will also give you the exact directions you need.
Once parked at the parking lot, you will start the hike at the trail beside the kiosk at the front of the parking lot entrance. Along the trail you will cross a wooden foot bridge and a small cascade of a waterfall. There is a small foot path to the right that people have made that takes you to the top of the waterfall. You will see it, when you start to descend, and the waterfall will be in your view on the right. That foot path doesn't loop anywhere or reconnect, so you'll need to retrace your footsteps back to the main trail to go to the bottom of the waterfall.
The trail ends at the waterfall and is about 2.3 of a mile.
This waterfall spot can be quite popular in the summertime with families, kids, and hikers. There isn't much of a swimming hole here, just some wading around you can do that's about knee-deep. It's a great place to bring the hammock, a book, your dog, or kids here to enjoy a day outdoors away from the city! Hike during the spring, and you'll see an abundance of different colored wildflowers!
ReviewsLeave a Review
Overall rating:
Chris Hickman
almost 2 years ago
Great short hike! The falls are very impressive
Caitlin Foster
about 2 years ago
Family Fun Hike
Fun short hike! Took my dog and 8 year old son for their first hike and they had a blast
April Shroff
about 3 years ago
Magical Place! A hidden gem!
We went to Stillhouse today for the first time, and wow! What a magical place! The beginning of the trail has a very whimsical/fairy vibe. It's an easy hike to a beautiful waterfall. We had the place to ourself and enjoyed wading and climbing the falls. Thanks for the tip!
Nearby Adventures
Hike to Rattlesnake Falls
1.4 miles 256 ft gain
Canoe down the Duck River
Backcountry Camp at the Wildcat Shelter
2.8 miles
Hike the Mossy Ridge Trail
4.5 miles


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The yeast Isw2 chromatin remodeling complex functions in parallel with the Sin3-Rpd3 histone deacetylase complex to repress early meiotic genes upon recruitment by Ume6p. For many of these genes, the effect of an isw2 mutation is partially masked by a functional Sin3-Rpd3 complex. To identify the full range of genes repressed or activated by these factors and uncover hidden targets of Isw2-dependent regulation, we performed full genome expression analyses using cDNA microarrays. We find that the Isw2 complex functions mainly in repression of transcription in a parallel pathway with the Sin3-Rpd3 complex. In addition to Ume6 target genes, we find that many Ume6-independent genes are derepressed in mutants lacking functional Isw2 and Sin3-Rpd3 complexes. Conversely, we find thatume6 mutants, but not isw2 sin3 or isw2 rpd3 double mutants, have reduced fidelity of mitotic chromosome segregation, suggesting that one or more functions of Ume6p are independent of Sin3-Rpd3 and Isw2 complexes. Chromatin structure analyses of two nonmeiotic genes reveals increased DNase I sensitivity within their regulatory regions in an isw2 mutant, as seen previously for one meiotic locus. These data suggest that the Isw2 complex functions at Ume6-dependent and -independent loci to create DNase I-inaccessible chromatin structure by regulating the positioning or placement of nucleosomes.
The regulation of RNA synthesis is a complex process affected by many factors that influence the initiation, elongation, and termination of transcription. These factors include transcriptional activators and repressors, as well as general transcription factors. In eukaryotes, the compaction of DNA into chromatin provides an additional level of complexity, due to the fact that chromatin structure is inhibitory to many protein-DNA interactions required for transcription (43). Consequently, chromatin structure must be dynamically regulated for many genes whose transcription is repressed or induced at different times of the cell cycle, at different phases of development, or by changes in growth conditions.
A large number of proteins from several organisms have been found to modify chromatin structure. These factors can be grouped into two classes based on their biochemical activities: histone-modifying enzymes and ATP-dependent chromatin remodeling complexes (for reviews, see references 29, 30, 36, 42, 45, 54, and55). Histone-modifying enzymes use several different enzymatic mechanisms to covalently modify histone proteins. These modifications include acetylation, phosphorylation, ubiquitination, and methylation. The second class of factors uses the energy of ATP hydrolysis to alter histone-DNA contacts, often leading to repositioning of histone octamers on DNA (56).
The role of histone acetylation in the regulation of transcription has been studied extensively (11, 42, 57). With some exceptions, high levels of histone acetylation correlate with transcriptionally active regions of chromatin, while lower levels of acetylation are found in transcriptionally inactive regions. Histone acetylation is regulated by the activities of histone acetyltransferase and histone deacetylase (HDAC) complexes, many of which function as transcriptional activators or repressors, respectively (42, 44, 45).
Three major families of HDACs have been conserved in eukaryotes, represented by the Rpd3, Hda1, and Sir2 proteins (12, 32). The RPD3 gene was first identified genetically in yeast as a transcriptional repressor of several genes (52, 53). It was later found that Rpd3 proteins in several species associate with Sin3p and other Sin3-associated proteins to form a multiprotein complex (1, 24, 59). Recruitment of this complex by transcriptional repressors leads to local deacetylation and repression of transcription (14, 15, 24, 26, 33, 37, 59).
A variety of ATP-dependent chromatin remodeling factors have also been identified from several organisms and can be grouped into three classes based on the ATPase subunit present in each complex: SWI/SNF, ISWI, and CHD1 (6, 22, 29, 38). Members of the ISWI class of chromatin remodeling factors were first identified biochemically inDrosophila, following their ATP-dependent activity in disrupting nucleosomes or imparting regular spacing on nucleosome arrays in vitro. These studies led to the identification of three ISWI-containing complexes, NURF (48, 50), CHRAC (51), and ACF (20). Subsequently, several other ISWI-containing complexes were identified in humans (3, 34, 35, 39), yeasts (49), and Xenopus(13, 28), based on sequence homology toDrosophila ISWI.
Although the biochemical activities of several ISWI complexes have been studied in detail, the in vivo functions of this class of factors are only beginning to be identified. The Drosophila ISWI gene is essential for development and cell viability (9). Drosophila ISWI mutants also have reduced expression of the Ubx and engrailedgenes, suggesting the requirement of one or more ISWI-containing complexes for the expression of these genes. In addition,ISWI mutants are compromised for male X chromosome integrity (9).
It was recently found that one of the two yeast ISWI complexes, the Isw2 complex, functions during vegetative growth to repress genes induced early in meiosis (10). Many early meiotic genes are repressed under these conditions by the Sin3-Rpd3 HDAC complex upon recruitment by the sequence-specific DNA-binding protein, Ume6p (24, 25, 40). Like the Sin3-Rpd3 complex, the Isw2 complex appears to be recruited to the promoters of early meiotic genes by Ume6p; however, repression by the Isw2 complex occurs independently of the Sin3-Rpd3 complex. Analysis of chromatin structure of one Ume6 target gene in wild-type and various mutant cells reveals that the Isw2 complex functions to create DNase I-inaccessible chromatin structure by altering the positions of nucleosomes upstream of the Ume6p-binding site. These data reveal that the Isw2 complex functions in parallel with the Sin3-Rpd3 complex to repress the transcription of common target genes.
While it is clear that Isw2 and Sin3-Rpd3 complexes collaborate to repress Ume6 target genes, it is not clear whether this collaboration extends to any Ume6-independent genes. To investigate this possibility, we compared the full genome transcriptional profiles of mutants defective in one or more of these factors. We also competed single mutants of sin3 and rpd3 with isw2 sin3 and isw2 rpd3 double mutants directly on microarray slides to identify genetic interactions between Isw2 and Sin3-Rpd3 complexes that are not always detectable in traditional mutant versus wild-type competitions. We find that while Ume6 target genes represent a major group of genes repressed by these complexes, a large number of Ume6-independent genes are derepressed in mutants defective in Sin3-Rpd3 and Isw2 complex functions. A comparison of isw2 and rpd3 deletion and catalytically inactive mutants reveals differences in both phenotype and transcriptional profiles which may result from functions of these proteins independent of their known catalytic activities, inhibition of related factors by catalytically inactive proteins, or a combination of the two. We also find that ume6 mutants have a reduced fidelity of chromosome segregation which results in higher rates of chromosome gains and losses relative to those in wild-type cells, suggesting a role for Ume6p in chromosome segregation that is independent of Isw2 and Sin3-Rpd3 complexes. Chromatin structure analyses of two Ume6-independent genes that require Sin3-Rpd3 and Isw2 complexes for proper regulation reveal that ISW2 function is required in both instances for the formation of DNase I-inaccessible chromatin structure, as previously observed for the Ume6 target geneREC104 (10). These data suggest that Sin3-Rpd3 and Isw2 complexes collaborate to repress the transcription of Ume6-dependent and some Ume6-independent genes and that the Isw2 complex functions to create DNase I-inaccessible chromatin structure at the promoters of many of these genes.
Strains.Unless indicated, all yeast strains were derived from W1588-4C. This strain is congenic to W303-1A, except that a weakrad5 mutation in the original strain W303 is repaired (60). Deletion and catalytically inactive mutations of theISW2, SIN3, RPD3, and UME6genes were described previously (10). The CFIII minichromosome (41) was introduced into isogenic wild-type and mutant strains by crossing an isw2 ume6double mutant (YTT1065) with SBY475 (courtesy of Sue Biggins, Fred Hutchinson Cancer Research Center). The colony sectoring assay for measuring rates of minichromosome loss was done as previously described (16, 58).
RNA isolation.RNA samples were prepared from mutant and wild-type cells grown at 30°C in YEPD medium (2% Bacto Peptone, 1% yeast extract, 2% glucose) to early log phase (optical density at 660 nm, 0.7) using acid phenol extraction. To ensure identical growth conditions, strains were grown in aliquots of media from a common preparation. mRNA was prepared using Oligotex beads (Qiagen) as described by the manufacturer.
Production of spotted microarrays.Microarray construction and hybridization protocols were modified from those described elsewhere (8). Yeast microarrays were constructed using a set of ∼6,200 open reading frame (ORF)-specific PCR primer pairs (Research Genetics, Huntsville, Ala.), which were used to amplify each ORF of the yeast genome. Individual PCR products were verified as unique via gel electrophoresis and purified using ArrayIt 96-well PCR purification kits (TeleChem International, Sunnyvale, Calif.). Purified PCR products in 3× SSC (1× SSC is 0.15 M sodium chloride plus 0.015 M sodium citrate [pH 7.0]) were mechanically spotted onto polylysine-coated microscope slides using an OmniGrid high-precision robotic gridder (GeneMachines, San Carlo, Calif.).
Microarray hybridizations and data analysis.The protocol used for cDNA labeling was a modification of a protocol described elsewhere (http://cmgm.stanford.edu/pbrown/protocols/aadUTPCouplingProcedure.htm ). Briefly, labeled cDNA targets were prepared by reverse transcription of 2 μg of mRNA using oligo(dT)18 primer in the presence of 0.2 mM 5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphate (Sigma-Aldrich Company, St. Louis, Mo.), 0.3 mM dTTP, and 0.5 mM each dATP, dCTP, and dGTP. Following cDNA synthesis, either Cy3 or Cy5 monoreactive fluor (Amersham Life Sciences, Arlington Heights, Ill.) was covalently coupled to the cDNA-incorporated aminoallyl linker in the presence of 50 mM sodium bicarbonate (pH 9.0). Two color expression profiles were generated using microarrays in which reference and experimental cDNA targets were labeled with different fluors. Following cohybridization to the chip, a fluorescent image of a microarray was collected at both emission wavelengths using a GenePix 4000 fluorescence scanner (Axon Instruments, Inc., Foster City, Calif.), and image analysis was performed using GenePix Pro microarray acquisition and analysis software.
Four microarray hybridizations were carried out for each comparison of mutant versus wild type or mutant versus mutant (two sets of two reverse fluor combinations). We carried out a Bayesian background correction, described in detail by Kooperberg et al. (31). Briefly, we assume that both the foreground and the background intensities of both channels for each spot come from a normal distribution with an unknown mean and variance. After estimating the variance parameters from the data, we can estimate for each channel the posterior distribution of the difference between the foreground and the background means as well as their ratio, which is the quantity of interest. To do this, we assume an uninformative uniform prior on the intensities. The effect of this procedure is that for genes where the intensities are high, the estimated ratio is very similar to the traditional estimate, but for spots where the foreground intensity is close to the background intensity, the estimate for the ratio is shrunk slightly toward one, yielding a substantial reduction in variance for these spots.
All calculations involving expression ratios were carried out on the (natural) log scale by computing averages and standard errors (SEs). Averages and SEs were then converted to ratios by assuming that the average of the log ratios follows the normal distribution (which seems reasonable given both the error distribution of log ratios and the central-limit theorem). The average ratio then has a log-normal distribution. This average and its SE are related, as described previously (21), to the average and the SE of the log ratios by the following equations: For analysis of the genomic locations of misregulated genes andcis element searches of promoter sequences, we used the GENESPRING software package (Silicon Genetics).
Chromatin structure analysis.Digestion of chromatin was performed with crude preparations of nuclei (17) as described previously (10). After digestion with micrococcal nuclease or DNase I, DNA was purified, digested withEcoRI and PstI (for POT1) orEcoRI and XbaI (for SUC2), and subjected to indirect end labeling. The probe used for indirect end labeling of the POT1 locus was a PCR product extending from +406 to +623 with respect to the initiation codon. The probe used for indirect end labeling of the SUC2 locus was a PCR product extending from −885 to −661 with respect to the initiation codon.
Overlapping functions of Isw2 and Sin3-Rpd3 complexes.Recently, it was found that the Isw2 complex functions during mitotic growth in Ume6p-dependent repression of early meiotic genes in a pathway parallel to that of the Sin3-Rpd3 HDAC complex (10). While it is clear that Sin3-Rpd3 and Isw2 complexes function in the repression of Ume6 target genes, it is not known whether this collaboration extends to genes not regulated by Ume6p. To determine the full extent to which Sin3-Rpd3 and Isw2 complexes function in parallel pathways of transcriptional repression, we used cDNA spotted microarrays representing >96% of all yeast genes to analyze the genome-wide expression profiles for mutants defective in one or both of these complexes. For each mutant, four independent mutant versus wild-type microarray hybridizations were carried out, and the average expression ratio and standard error for each spot were determined (see Materials and Methods for details; the full data set can be obtained atftp://milano.fhcrc.org/ArrayLab/Fazzio/ ). Initially, we focused on isw2, rpd3, and sin3 single null mutants, as well as isw2 rpd3 andisw2 sin3 double null mutants. In addition, we analyzed expression profiles for ume6 and isw2 ume6 null mutants; these data are discussed below. We observed relatively few changes in transcript levels for theisw2 single mutant, as previously described for anisw2/isw2 homozygous mutant diploid strain (18). Because it was previously found that Isw2 and Sin3-Rpd3 complexes can each partially compensate for the lack of the other in repression of common target genes, we focused on genes that require defects in both complexes for moderate levels of derepression. As shown in Table 1, larger numbers of genes are moderately derepressed (>3-fold) in isw2 rpd3 and isw2 sin3 double mutants than in single mutants. Similarly, more genes are derepressed to higher levels (>5-fold, >10-fold) in double mutants than in single mutants, revealing that Sin3-Rpd3 and Isw2 complexes function in parallel pathways to repress the transcription of a considerable group of genes.
As expected, many genes found to be repressed by Sin3-Rpd3 and Isw2 complexes include known targets of Ume6p and genes predicted to be Ume6 targets by virtue of an upstream Ume6p-binding site (URS1) (Table1). We also searched for a second Ume6-binding sequence previously identified in the promoter of the PHR1 gene (46); however, this sequence was not overrepresented in the promoters of genes derepressed in any of our mutants. In addition to genes repressed by Ume6p, we observed many genes that require Sin3-Rpd3 and Isw2 complexes for repression that lack URS1 sequences. The majority (82%) of these genes are not substantially derepressed in the ume6 mutant, suggesting that derepression inisw2 sin3 and isw2 rpd3mutants is not a secondary consequence of the upregulation of early meiotic genes (see the full data set for details). Together, these data reveal that a substantial group of genes requires Sin3-Rpd3 and Isw2 complexes for repression, independent of Ume6p.
In addition to the role of the Isw2 complex in repression, we also found that the transcription of a small number of genes is reduced in the isw2 mutant (see the full data set for details). For these genes, the relationship between the Isw2 complex and Sin3-Rpd3 complex in the activation or maintenance of basal transcription is less clear. Few genes have significantly reduced expression in theisw2 deletion mutant (zero genes reduced >3-fold and one gene reduced >2-fold). In addition, many genes with reduced expression in the sin3 or rpd3 single mutant are unaffected or oppositely affected by the addition of an isw2 mutation in these backgrounds. These data suggest a relatively minor role of the Isw2 complex in the transcriptional activation or maintenance of basal transcription under the conditions used (logarithmic growth in rich glucose medium).
Hidden functions of the Isw2 complex revealed insin3 and rpd3 mutant backgrounds.The data in Table 1 suggest that many targets of Isw2-mediated repression may be masked in isw2 mutant cells by the presence of a functional Sin3-Rpd3 complex. To identify ISW2target genes affected in this way, we performed two experiments. First, we divided isw2 rpd3/wild-type expression ratios by rpd3/wild-type ratios for all genes (Table2); similarly, isw2 sin3/wild-type expression ratios were divided bysin3/wild-type ratios (Table 2). In theory, the ratios obtained through these calculations should reveal defects in transcriptional regulation resulting from the loss of Isw2 function in an rpd3 or sin3 mutant background. However, when we compared these data to those obtained by Northern blotting (Table2), we noticed several inconsistencies. For several meiotic genes previously found to be targets of Isw2-dependent repression, the ratios derived from one or both calculations described above do not reflect known derepression due to the isw2 mutation (cf.HOP1, SPO11, REC104, andSPO1). Under the growth conditions used for this experiment (logarithmic growth of haploid cells in rich glucose medium), many genes are tightly repressed in wild-type cells. In microarray experiments, expression ratios for genes with a low signal in the wild-type channel are often underestimated relative to ratios measured by Northern blotting (31). As a result, isw2 rpd3/ wild-type expression ratios for these genes measured using microarrays can be similar to rpd3/wild-type ratios; likewise, isw2 sin3/wild-type expression ratios can be similar to sin3/wild-type ratios. Consequently, when double mutants are divided by single mutants to compare their expression profiles, some differences observed by Northern blotting are not revealed.
To circumvent this problem, we measured isw2 rpd3/rpd3 ratios and isw2 sin3/sin3 ratios directly by labeling single- and double-mutant RNA samples with opposing dyes and hybridizing them to the same microarray slide. In contrast to the typical method of measuring mutant/wild-type ratios, this approach uses rpd3or sin3 single mutants as "references" against which double-mutant expression levels are compared. As a result, the problem of underestimated expression ratios resulting from minimal wild-type expression is significantly reduced for genes derepressed inrpd3 or sin3 mutants. For both theisw2 rpd3-rpd3 and the isw2 sin3-sin3 comparisons, the expression ratio obtained from the direct (mutant-versus-mutant) hybridization is higher for most genes examined than that obtained by dividing two different mutant/wild-type expression ratios (Table 2). In addition, when expression ratios obtained by direct hybridization are compared to those calculated from mutant–versus–wild-type experiments, we find that direct hybridization more accurately detects derepression observed by Northern blotting. While the values of expression ratios measured for mutant-versus-mutant experiments were not always identical to those calculated from Northern data, this method qualitatively identified derepression due to the isw2 mutation for all 10 genes analyzed in both rpd3 and sin3 mutant backgrounds.
In an attempt to categorize the targets of Isw2-dependent repression, we analyzed more closely the set of genes identified above that are derepressed in isw2 rpd3 or isw2 sin3 double mutants relative to sin3 orrpd3 single mutants. This class of genes requires the Isw2 complex for repression in the absence of a functional Sin3-Rpd3 complex. We focused on the set of genes derepressed at least 1.7-fold in one or both of the direct hybridization experiments, as this cutoff level allowed for inclusion of ∼90% of genes known to be repressed by the Isw2 complex (as measured by Northern blotting) (Table 2) but did not include any genes found by Northern blotting to be unaffected in isw2 mutant cells (data not shown). By these criteria, 315 genes (∼5% of all yeast genes) belonging to many different functional categories were found to be derepressed when anisw2 mutation was present in rpd3 and/orsin3 mutant backgrounds; representatives of this group of genes are listed in Table 3. For comparison, only 112 genes met this 1.7-fold threshold in the isw2 single mutant. As expected, a significant portion (∼20%) of the 315 genes in this group contained the core Ume6p-binding site, 5′-GGCGGC-3′, in the 500 bp upstream of their initiation codons; the majority (∼72%) of these genes were also found to be derepressed in ume6 andisw2 ume6 mutant backgrounds (see the full data set). No other sequences were significantly overrepresented in the promoters of these genes. We also observed 80 genes whose expression is decreased more than 1.7-fold in one or both double mutants relative torpd3 or sin3 single mutants, consistent with a lesser role for the Isw2 complex in the activation of transcription. The majority of these genes are uncharacterized ORFs, and no functional category of genes is highly represented in this group.
Catalytically inactive isw2 and rpd3mutants differ from deletion mutants in phenotype and transcriptional profiles.Isw2p has nucleosome-stimulated ATPase activity that is required for both chromatin remodeling in vitro (49) and repression of early meiotic genes in vivo (10). Similarly, HDAC activity of Rpd3p is required for normal levels of repression of target genes (7, 10, 23). However, several deacetylase-defective mutants of rpd3 were previously found to be only partially defective in the repression of a LexA reporter construct (23), suggesting one or more functions of Rpd3p that are independent of its deacetylase activity. In addition, overexpression of catalytically inactive Rpd3 protein in wild-type cells results in a partial dominant-negative phenotype (23). It is therefore possible that catalytically inactive mutants of isw2 and rpd3 exhibit defects in transcriptional regulation not seen in deletion mutants. In deletion mutants, related chromatin remodeling factors may partially compensate for the deleted proteins, whereas catalytically inactive proteins may prevent these factors from accessing chromatin. In addition, accessory proteins shared by multiple chromatin remodeling factors may be titrated by catalytically inactive proteins. Alternatively, deletion mutants may have defects in transcriptional regulation not observed in catalytically inactive mutants due to functions of these proteins that are independent of their known catalytic activities.
To determine the differences between catalytically inactive and deletion mutants of isw2 and rpd3, we analyzed the transcriptional profiles of isw2 and rpd3single catalytically inactive mutants as well as the isw2 rpd3 double catalytically inactive mutant. For this purpose, we analyzed an rpd3 mutation by which a conserved histidine residue at position 151 is changed to alanine; this mutation was previously found to eliminate the deacetylase activity of Rpd3p (23). Similarly, we analyzed a previously characterizedisw2 substitution mutation (which changes lysine 214 to arginine), known to eliminate the ATPase activity of Isw2p.
While many of the same genes were misregulated in deletion and catalytically inactive mutants of isw2 and rpd3, in each instance there were considerable differences in transcriptional profiles between the two types of mutants (Fig.1A). While relatively few genes are derepressed at least twofold in either isw2 mutant, a substantial amount of nonoverlap is apparent for the two transcriptional profiles. A moderate number of genes are derepressed at least twofold in the rpd3 deletion mutant but not in the catalytically inactive mutant. More strikingly, a large number of genes that are derepressed at least twofold in the rpd3catalytically inactive mutant do not meet this threshold in the deletion mutant (Fig. 1A, right panel). Most of these genes are not significantly derepressed in sin3 mutant cells, suggesting that Rpd3p is required for the repression of this group of genes independent of Sin3p. In contrast, most genes derepressed in thesin3 mutant are also derepressed in one or bothrpd3 mutants, with the largest single group ofsin3-responsive genes being those derepressed in all three mutants. These data suggest that Sin3p acts primarily in conjunction with Rpd3p to repress the transcription of common target genes, whereas Rpd3p appears to be required for the repression of some genes independent of Sin3p.
To further investigate the differences in transcriptional profiles between deletion and catalytically inactive mutants of rpd3, we searched the 489 genes derepressed in the catalytically inactive mutant but not in the deletion mutant for common sequences within their presumed regulatory regions (within 500 bp upstream of their initiation codons). While we did not find any known regulatory elements, several elements with overlapping sequences were overrepresented in this group to a high level of significance (Fig. 1B). The consensus for these sequences, 5′-GNGATGAGNT-3′, is present in the upstream 500 bp of 174 yeast genes. Of these, 56 (32%) are derepressed at least 2-fold and 131 (75%) are derepressed at least 1.5-fold in therpd3 catalytically inactive mutant. This sequence was previously identified by two independent computational approaches and found to be overrepresented in the regulatory regions of a group of genes with a common expression pattern during the cell cycle (5, 47). These data cannot distinguish whether this sequence functions directly in Rpd3-mediated repression or whether therpd3 catalytically inactive mutation indirectly leads to derepression of these genes. However, the fact that this sequence is overrepresented in the regulatory regions of genes derepressed in the same mutant as well as coexpressed during the cell cycle suggests that it may function in transcriptional regulation. Among the genes that contain this sequence in their upstream regulatory regions are those encoding components of the RNA processing and degradation machinery, as well as subunits of RNA polymerases I and III (47) (data not shown). This element may therefore coordinate the regulation of genes involved in several different aspects of RNA metabolism.
Consistent with the differences in transcriptional profiles, we also observed differences in synthetic growth phenotypes between deletion and catalytically inactive mutants of isw2 andrpd3. It was previously found that the isw2 rpd3 double deletion mutant exhibits a slow-growth phenotype at 30°C and fails to form colonies at 37°C (10). In contrast, the double catalytically inactive mutant grows at a rate similar to that of the wild type at 30°C and grows slightly more slowly than the wild type at 37°C (Fig. 1C). We also find that the rpd3 single deletion mutant grows slightly more slowly than the wild type at 37°C, while growth of the single catalytically inactive mutant is unaffected. These results suggest that the isw2 rpd3 double deletion mutant is impaired in one or more functions required for cell growth or division and that this defect is enhanced at higher temperatures. The phenotype of the double catalytically inactive mutant suggests that it is less severely impaired in these functions. The partial growth defect of the double catalytically inactive mutant at 37°C is very similar to that observed previously for an isw2 sin3double mutant (10). These data, combined with the transcriptional data discussed above, suggest one or more functions of Rpd3p that require neither deacetylase activity nor Sin3p. However, the fact that many more genes are derepressed in the rpd3catalytically inactive mutant than in the deletion mutant suggests that the catalytically inactive protein may inhibit other transcription or chromatin remodeling factors. The differences in growth phenotypes and transcriptional profiles between deletion and catalytically inactive mutants may therefore result from a combination of these two effects.
Genomic instability of ume6 mutants.The Isw2 complex is recruited by Ume6p and functions in repression of genes that contain the Ume6p-binding site, URS1 (10). Since repression of early meiotic genes by Isw2 and Sin3-Rpd3 complexes is fully dependent on Ume6p, we wished to determine whetherUME6 function is required for repression of nonmeiotic genes by these complexes. To determine the extent to which Isw2 and Sin3-Rpd3 complexes depend on Ume6p, as well as the extent to which Ume6p functions through these complexes, we analyzed the expression profiles for ume6 and isw2 ume6 mutants.
Surprisingly, more genes were derepressed to high levels inume6 and isw2 ume6 mutants than inisw2 rpd3 or isw2 sin3mutants (Table 1), suggesting the presence of a number of Ume6 targets that are not regulated by Isw2 and Sin3-Rpd3 complexes. In addition, we found that many genes with no nearby URS1 sequence were derepressed inume6 and isw2 ume6 mutants (Table 1). Upon closer inspection, a much higher-than-average density of derepressed genes was located on chromosome 16 (for the ume6mutant) or chromosomes 9 and 16 (for the isw2 ume6 double mutant) (data not shown). Recently, Hughes et al. observed similar chromosomal expression biases in ∼8% of a large number of expression studies (19). This group examined the nature of the expression biases for each of these mutants and found, for nearly every one examined, that the biases resulted from duplications or deletions of chromosomal segments or whole chromosomes.
To determine whether ume6 and isw2 ume6 mutants contained chromosomal duplications, we isolated DNA from mutant and wild-type cells, labeled each, and performed competitive hybridizations on microarray slides as described previously (19). We analyzed the DNA contents of two independent ume6 mutants and three independentisw2 ume6 mutants constructed in two different strain backgrounds in our laboratory, as well as one ume6mutant obtained from another laboratory (Table4). For all of the mutants, we found evidence that at least one and sometimes two or more chromosomes were duplicated. During the course of this work, independent expression data for a ume6 mutant were published (2); analysis of these data reveals chromosomal expression biases that strongly suggest the presence of chromosomal duplications (data not shown), supporting our findings. Independent DNA isolates from the sameume6 mutant revealed variability in which chromosomes were duplicated, suggesting that duplicated chromosomes were not always maintained during growth (Table 4). While the most common duplications were of chromosomes 9 and 16, we also observed, less frequently, duplications of chromosomes 1, 2, 3, and 8. Hughes et al. (19) found that for several mutants, the duplicated chromosomes or chromosomal segments contained genes homologous to the mutated gene, suggesting that selection for extra copies of homologous genes might partially compensate for the lack of the mutated gene. However, for UME6, we have found no close homolog within the yeast genome. In addition, the high degree of variability in chromosomes duplicated in ume6 mutants argues against the possibility that these mutants are selected for extra copies of particular genes.
Alternatively, chromosomal duplications in ume6 mutants may result from a general defect in chromosome segregation. In this scenario, chromosome loss would be predicted to occur in addition to chromosomal duplications. However, in our expression studies, we would only have observed chromosomal duplications, since chromosome loss is lethal to haploid cells. To test this possibility, we constructed strains containing a minichromosome harboring the SUP11gene, which suppresses the ade2-101 mutation in our yeast strains, rendering colonies white. Because this chromosome is dispensable for cell viability, chromosome loss rates can easily be measured by monitoring differences in colony color after nonselective growth (16, 41). With this assay, we found thatume6 and isw2 ume6 mutant cells lose the minichromosome roughly 26- to 62-fold more often than wild-type cells (Table 5). These results suggest that chromosomal duplications observed in ume6 mutants result from chromosome missegregation events.
The Isw2 complex is required for the formation of DNase I-inaccessible chromatin structure at two Ume6-independent loci.Chromatin structure analysis of one gene (REC104) targeted for repression by Isw2 and Sin3-Rpd3 complexes via Ume6p revealed that the Isw2 complex forms DNase I-inaccessible chromatin structure upstream of the Ume6p-binding site (URS1) (10). Inisw2 mutants, increased accessibility of chromatin near the URS1 sequence appeared to be due to a shift in the positions of two or three nucleosomes directly upstream of this site. The formation of an inaccessible chromatin structure at this site by the Isw2 complex requires a functional UME6 gene but is unaffected by mutation of the RPD3 gene.
Because the formation of DNase I-inaccessible chromatin structure by the Isw2 complex occurs directly adjacent to the Ume6p binding site, it is possible that Ume6p modifies or regulates the activity of the Isw2 complex upon recruitment, in a manner unique to Ume6 target genes. If this theory is correct, the Isw2 complex may modify chromatin structure differently at loci where it functions independently of Ume6p. Alternatively, the Isw2 complex may function similarly to create inaccessible chromatin structure at all genes for which it regulates chromatin structure, regardless of the mechanism of recruitment. To distinguish between these possibilities, we analyzed the chromatin structure of the POT1 gene using micrococcal nuclease and DNase I digestions of chromatin, followed by indirect end labeling (Fig. 2A). The POT1 gene was selected because it is repressed in parallel pathways by Isw2 and Sin3-Rpd3 complexes, yet data from both microarray and Northern blotting experiments confirmed little or no change inPOT1 expression in a ume6 mutant (Fig. 2A) (see the full microarray data set). In addition, no Ume6p-binding site (URS1) is present in the POT1 upstream regulatory region. As previously observed for the REC104 gene, the positions of three nucleosomes at the POT1 locus appear to change in theisw2 mutant, while no change is observed in therpd3 mutant. As with the REC104 gene, these changes are accompanied by an increase in DNase I hypersensitivity near the promoter. In contrast to the situation for the REC104locus, chromatin structure changes at the POT1 locus extend well into the coding region in isw2 mutant cells. Nevertheless, DNase I-inaccessible chromatin structure established at the POT1 locus in the presence of the Isw2 complex is very similar to chromatin structure at the REC104 locus. These data suggest that the Isw2 complex may function similarly to remodel chromatin at these two loci, despite the fact that repression ofREC104 by the Isw2 complex requires UME6function, while repression of POT1 transcription isUME6 independent.
Although the Isw2 complex appears to have only a small role in transcriptional activation or maintenance of basal transcription, we found that transcription of one gene (SUC2) was significantly reduced in isw2, sin3,rpd3, isw2 sin3, and isw2 rpd3 mutants (see the full data set). The level of expression of SUC2 was lower in isw2 sin3 and isw2 rpd3 double mutants than in any single mutant, indicating that Isw2 and Sin3-Rpd3 complexes affect SUC2 transcription independently. These data were confirmed by Northern blotting (Fig. 2B, Relative expression). Under the conditions of RNA isolation used for these studies (2% glucose media), the SUC2 gene is actively repressed. Therefore, our results suggest that Isw2 and Sin3-Rpd3 complexes are required for low levels of basal SUC2 transcription. The chromatin structure of the SUC2 promoter has been studied extensively, revealing a role for the Swi-Snf complex in the creation of nuclease-accessible chromatin during transcriptional activation (17). Since increased transcription is often associated with a more accessible chromatin structure, it is possible that the Isw2 complex also functions to make chromatin more accessible at the SUC2locus, in contrast to its functions to create DNase I-inaccessible chromatin structure at the REC104 and POT1 loci.
To test this possibility, we probed the chromatin structure of theSUC2 locus using DNase I digestions of chromatin, followed by indirect end labeling (Fig. 2B). The DNase I digestion patterns of wild-type chromatin and isw2 mutant chromatin are very similar for the SUC2 ORF and much of the upstream regulatory region. However, a notable increase in DNase I cleavage is observed approximately 500 bp upstream of the initiation codon in theisw2 mutant. As previously found for the REC104and POT1 loci, the DNase I digestion pattern ofrpd3 mutant chromatin was very similar to that of wild-type chromatin. Thus, at the SUC2 locus, as at theREC104 and POT1 loci, the Isw2 complex, but not the Sin3-Rpd3 complex, is required for the creation of DNase I-inaccessible chromatin structure. However, unlike the situation for the REC104 and POT1 genes, the increase in DNase I accessibility is associated with a decrease in SUC2transcription in the isw2 mutant. These data suggest that the Isw2 complex functions by a common mechanism at Ume6-dependent and -independent loci to create DNase I-inaccessible chromatin structure and that ISW2-dependent inaccessible chromatin structure can affect transcription both positively and negatively.
Because it was previously found that the functional Sin3-Rpd3 complex could often compensate for the loss of Isw2 function in repression of common target genes, we compared the transcriptional profiles of mutants defective in one or both complexes to determine the full extent of their overlap. In addition, we wished to determine the extent to which the repressive functions of Isw2 and Sin3-Rpd3 complexes depend on Ume6p. Our data indicate four main conclusions. First, the Isw2 complex functions in a pathway parallel to that of the Sin3-Rpd3 complex to repress transcription of a substantial number of genes. We find that synthetic phenotypes (those observed only when anisw2 mutation is combined with a sin3 orrpd3 mutation) are more consistently revealed using double mutant versus single mutant hybridizations, especially for genes that are tightly repressed in wild-type cells. While the largest single group of genes repressed by these complexes consists of Ume6 target genes, the majority of genes that require Isw2 and Sin3-Rpd3 complexes for repression are Ume6 independent.
Second, chromatin structure analyses suggest that the Isw2 complex is required to create DNase I-inaccessible chromatin structure in the upstream regulatory regions of two Ume6-independent genes. At this time, we cannot exclude the possibility that the observed changes in chromatin structure and transcription are due to indirect effects ofisw2 mutation, since we have been unable to localize the Isw2 complex to specific chromosomal loci. The rpd3 mutant is defective in regulation of the POT1 and SUC2genes to a similar extent as the isw2 mutant. However, only cells with the isw2 mutation show the mutant nuclease digestion patterns at these loci, arguing against the possibility that chromatin structure defects in isw2 mutant cells result from misregulation of transcription. Because SUC2 transcription is decreased in isw2 mutant cells, it appears that Isw2-dependent DNase I-inaccessible chromatin structure can have different effects on transcription at different loci. While it is not clear why inaccessible chromatin structure results in increased transcription of the SUC2 gene, one possibility is that it partially inhibits the binding of a transcriptional repressor ofSUC2. Consistent with this possibility, a binding site for the Mig1 repressor is located very close (at −499 with respect to the initiation codon) (4) to the DNase I-hypersensitive site present only in isw2 mutant cells.
Recently, Kent et al. showed that the Isw2 complex is required for the formation of wild-type chromatin structure upstream of three randomly selected Ume6-independent genes, FIG1, MET17, andPHO3, as revealed by differences in their MNase cleavage patterns in isw2 mutant cells relative to wild-type cells (27). However, transcription of the FIG1 andPHO3 genes is unaffected by the isw2 mutation, and MET17 transcription is only slightly increased inisw2 mutant cells (see the full data set). It is therefore possible that regulation of chromatin structure at these loci by the Isw2 complex serves a function other than transcriptional regulation. Alternatively, additional chromatin remodeling factors may function in parallel to regulate the transcription of these genes. In this case, loss of Isw2 function may be masked by these parallel factors, analogous to the relationship between Isw2 and Sin3-Rpd3 complexes in the repression of some early meiotic genes. Taken together, the data suggest that the Isw2 complex functions by a common mechanism at Ume6-dependent and -independent loci to create nuclease-inaccessible chromatin structure and that the ISW2-dependent inaccessible chromatin structure can affect transcription positively, negatively, or not at all, depending on the specific context.
Third, there are considerable differences in transcriptional profiles between deletion and catalytically inactive mutants of isw2and rpd3. These differences are most evident forrpd3 mutants, suggesting the presence of deacetylase-independent and Sin3-independent functions of Rpd3p, as well as the possibility that catalytically inactive Rpd3p may inhibit other repressors of transcription. Consistent with these differences,rpd3 deletion mutations show a synthetic growth defect in anisw2 mutant background, whereas catalytically inactive mutations of rpd3 and deletion mutations of sin3show less severe synthetic growth defects in this background. In contrast, a recently published comparison of the transcriptional profiles of rpd3 and sin3 deletion mutants suggests that virtually all Rpd3p functions require Sin3p (2). The major difference between this report and our data is our finding that a large number of genes require RPD3 but not SIN3 for repression. These conflicting conclusions may be explained by differences in strain background or conditions used for the growth of yeast cells. However, we previously observed phenotypic differences between rpd3 and sin3 deletion mutants in two different yeast strain backgrounds with regard to their synthetic growth defect in an isw2 mutant background (10). These data support the conclusion that Rpd3 has some Sin3-independent functions in vivo.
Fourth, Ume6p has one or more roles in chromosome segregation during haploid mitotic growth that are independent of Isw2 and Sin3-Rpd3 complexes. As a result, haploid ume6 mutants tend to accumulate chromosomal duplications. For this reason, expression data for the ume6 and isw2 ume6 mutants should be interpreted with caution, since chromosomal duplications result in artificial inflation of genes on the duplicated chromosomes and may indirectly affect the expression of additional genes. Despite this fact, we find that a large group of genes appears to be regulated by Isw2 and Sin3-Rpd3 complexes independently of Ume6p. It is noteworthy that the expression profiles for sin3,rpd3, isw2 sin3, and isw2 rpd3 mutants show no signs of chromosomal duplications in these mutants. In contrast, Hughes et al. previously found chromosomal duplications associated with rpd3/rpd3 andsin3/sin3 homozygous mutant diploids (19). These differences may result from differences in strain background or growth media or possibly from different effects of sin3 andrpd3 mutations in haploid and diploid cells. Nevertheless, our data suggest that Ume6p has one or more functions in chromosome segregation during haploid mitotic growth that are independent of Sin3-Rpd3 and Isw2 complexes under the conditions tested. It is possible that Ume6p regulates the transcription of specific genes independently of Sin3-Rpd3 and Isw2 complexes. Alternatively, Ume6p may function more directly in some aspect of chromosome segregation. The bias observed for duplications of chromosomes 9 and 16 may reflect a growth advantage associated with extra copies of these chromosomes inume6 mutant cells. However, it is also possible that these chromosomes are more susceptible than others to missegregation inume6 mutants.
We thank Sue Biggins, Marnie Gelbart, Mark Groudine, Cedar McKay, and Jay Vary for helpful discussions and critical reading of the manuscript. We also thank Sue Biggins and Randy Strich for yeast strains and Christine Jasoni for help with data processing.
This work was supported by a Pew Charitable Trust Biomedical Scholars fellowship and NIH grant GM58465 to T.T. The FHCRC DNA Array Facility is funded in part by NCI Cancer Center support grant 5P30 CA15704-28. T.G.F. is supported by a predoctoral fellowship from HHMI. C.K. is supported by NIH grant CA74841.
- Received 9 May 2001.
- Returned for modification 19 June 2001.
- Accepted 27 June 2001.
- Copyright © 2001 American Society for Microbiology


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This department offers an undergraduate major under the bachelor of science degree program. The basic requirements of this curriculum include: two years of chemistry, one year of physics, one year of general biology, one semester of calculus, and biostatistics. The major requirements include: one year of general genetics, introductory biochemistry, laboratory experience, and 12 credits of genetics and genetics-related courses chosen from an approved list.
Genetics is a bachelor's program for students seeking to understand how genes shape life, from fundamental cellular functions to population dynamics, and for students preparing to apply genetic and genomic concepts in such areas as medicine, biotechnology, biomedical research, agriculture, journalism, and public policy.
Advances in genome sequencing, bioinformatics, and our ability to manipulate the DNA of many organisms, including humans, have brought genetics to the forefront of many issues facing our society. These advances drive the growing need for health care providers, scientists and other professionals with a strong foundation in genetic and genomic analysis. Through coursework and diverse research opportunities, genetics majors gain broad insight into inheritance, gene function, genome organization, evolution, cutting-edge genetic technologies and therapies, and more.
A B.S. degree with a major in genetics positions students for many jobs in the biotechnology industry. Genetics majors are well prepared to pursue research-focused Ph.D. programs that provide further training for careers in biomedical and agricultural research. Genetics majors are highly competitive for admission to top medical schools, where there is a growing focus on personalized medicine, and genetic counseling programs.
Doebley, John (chair); Gasch, Audrey; Ikeda, Aki; Laughon, Al; Masson, Patrick; Payseur, Bret; Pelegri, Francisco; Perna, Nicole; Prolla, Tom; Schwartz, David; Skop, Ahna; Wassarman, David; Yin, Jerry
Associate Professors
Chang, Qiang; Hittinger, Chris; Pool, John
Assistant Professors
Sharp, Nathaniel; Werling, Donna; Zhong, Xuehua
Faculty Associates;
Tilmann, Kit; Vermillion Kalmon, Katie; Loewen, Carin
Undergraduate Advisors
Tilmann, Kit; Vermillion Kalmon, Katie; Loewen, Carin; Foley, Lauren


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Alexander FossåOncologistRadiumhospitaletOslo University Hospital
B-cell lymphoblastic leukemia/lymphoma is treated using ALL protocols where use of radiation therapy is often applied. For many patients, radiation therapy is not indicated. Radiation therapy is relevant for CNS manifestations in combination with other CNS-directed treatment or as part of CNS prophylaxis for certain subgroups of patients. Radiation therapy for testicular involvement prevents local recurrence. Total body irradiation may be used for certain patients who are candidates for stem cell transplantation. Whether radiation therapy is given to areas of B-lymphoblastic lymphoma with initial bulk and whether residual lesions are irradiated (with CR in bone marrow) is considered on an individual basis.
Radiation therapy alone is considered potentially curative only for localized stages of indolent lymphomas (stage I/PeI and localized stage II1/PeII1). Radiation therapy is generally given to the involved lymph node region/involved organ (involved field). At Oslo University Hospital, the nearest draining uninvolved lymph node region is included as part of this.
The involved area of the lymph node region includes visible tumor (GTV) with at least 1 cm margin to CTV in the transversal plane. In the craniocaudal direction, there should be at least 2 cm to CTV and CTV should generally include the involved lymph node region in the transversal plane. ITV is generated based on internal movement of CTV. The field borders are then around 3-5 cm from the macrotumor in the craniocaudal direction and at least 2 cm from the macrotumor in the transversal plane. For extranodal involvement (for example the eye, salivary glands, stomach, skin), the definition of target volume may vary from organ to organ. If the nearest draining lymph node region or parts of this are included, this is modeled in CTV.
Direct modeling on the simulator follows equivalent guidelines for determination of field borders. The margin for ITV to field border is 1-1.5 cm for setup uncertainty and and penumbra.
Standard fractionation for most patients is 2 Gy x 15.
If the disease is in a localized stage, radiotherapy to the involved lymph node area is recommended and possibly including the nearest draining neighboring region. Modeling and contouring follows the same principles as for indolent lymphomas.
Fractionation is 2 Gy x 15.
This diagnosis is made from a bone biopsy or aspirate dominated by mature, partially atypical plasma cells, and makes up 5% of plasma cell neoplasias. The presence of lytic solitary destruction of bone is common and evident by X-ray. Certain patients may have monoclonal gammopathy at serum protein electrophoresis. Myeloma should be excluded by standard examinations.
Treatment consists of curative radiation therapy. The prognosis is relatively good, but over half of patients develop generalized disease (myeloma) within 5-10 years after treatment.
Using 3D dose-planned treatment, the visible tumor is GTV. There is often soft tissue spreading outside the bone, therefore adequate imaging is necessary (CT or MRI) for evaluation. CTV should be modeled with at least 1 cm margin to GTV. If there is tumor involvement in vertebrae, all involved vertebrae and transverse processes should be included in CTV, with field borders lokated on the vertebral discs including one uninvolved vertebrae above and one below in the radiation field. The border to CTV in long bones should be 2-3 cm lengthwise in the marrow to be treated. Additional margin to ITV depends on the localization of the tumor, for example the thoracic wall.
Total dose and fractionation are normally 40 Gy as 2 Gy x 20. Data show that 59 Gy in fractions of 2 Gy gives a high rate of local control.
This diagnosis is made by a histological examination of an extraosseous neoplasm and constitutes 3-5% of plasma cell neoplasias. This disease is mainly (about 80%) localized to the upper respiratory tract. Some patients may have monoclonal gammopathy. Treatment consists of radiotherapy, and the prognosis is very good since few patients develop generalized disease.
With 3D dose-planned treatment, the visible tumor is GTV. CTV should be modeled with at least a 1 cm margin. An additional margin to ITV depends on the localization of the tumor. For tuning on the simulator, the field borders are located 2-3 cm from the macrotumor. It has been discussed in the literature whether one draining regional lymph node should be included, but Oslo University Hospital has not done this routinely in recent years. (Thomas Broe Christensen, Århus)
Total dose and fractionation are normally 40 Gy given as 2 Gy per fraction. Data suggest that this treatment gives a high rate of local control.
These lymphomas often occur as extranodal lymphomas and are often confined at the time of diagnosis. Marginal zone lymphomas are considered very radiosensitive. Radiotherapy for this diagnosis follows the same principles as for indolent lymphomas, but extranodal forms may be technically challenging to treat (such as stomach lymphomas).
Total dose and fractionation is 2 Gy x 15.
Less than 1/3 of patients are in stage I or II when diagnosed. Localized disease, or potentially localized disease rather, can be defined as stage I and stage II1 where only two neighboring lymph node regions are involved, which can be included in a suitable radiation field.
Treatment according to the Nordic lymphoma group's recommendation (www.nordic-lymphoma.org) is local radiotherapy with curative intention.
The involved area of the lymph node region includes visible tumor (GTV) with at least 1 cm margin to CTV. In the craniocaudal direction, (direction of lymph drainage) there should be at least 2 cm to CTV (3 cm in according to Nordic Lymphoma group's guidelines) and CTV normally includes all of the involved lymph node region in the transversal plane. ITV is generated based on assumed internal patient movement of CTV. Field limits will then be 3-5 cm from the macrotumor in the craniocaudal direction and at least 2 cm from the macrotumor in the transversal plane. For extranodal involvement, such as the eye, salivary glands, stomach, or skin, the definition of target volume will vary from organ to organ. If the nearest draining lymph node region or parts of this are included, this is modeled in the CTV. There is no convincing data to indicate extending the fields beyond this.
For direct modeling on the simulator, equivalent guidelines are followed for choice of field borders with margins 1-1.5 cm for setup error and penumbra.
The recommended total dose and fractionation are 30 Gy given as 2 Gy x 15.
Less than half of patients will develop recurrence outside the radiation field and systemic treatment is appropriate when necessary.
Radiation therapy is limited for stages II2-IV. In multiple palliative situations, radiotherapy may be a good alternative for tumor masses that threaten organ function such as obstruction of the bronchial tree, ureters, spinal compression, or venous stasis, or those causing pain. It is important to consider radiotherapy also in patients with advanced, chemo-resistant disease. Effects can be achieved with much lower doses than those usually applied for follicular lymphomas. A common fractionated regimen for palliative treatment of large fields is 2 Gy x 2. Radiotherapy is considered for some patients with residual lesions after effective chemotherapy such as patients with remaining transformed follicular lymphomas after instensive chemotherapy/HMAS.
Follicular lymphomas grade B are treated as DLBCL, and radiotherapy is used in combination with chemotherapy as for DLBCL.
Many studies that support the use of radiotherapy for aggressive B-cell lymphomas are antiquated and do not use current classification or systemic treatment with combined immuno-chemotherapy. For DLBCL, the guidelines are relatively well documented by some randomized studies, while for other lymphomas, guidelines are based more on analogical assessments.
Patients in stages I/PeI and patients with localized stages II (II1/PeII1) are considered for treatment analogous to strategies for early stages of DLBCL. Whether radiotherapy alone should be recommended is less certain, if so 2 Gy x 20 is recommended as for follicular lymphomas.
For localized disease (stage I/PeI and stage II1/PeII1) 3–6 CHOP21 + rituximab cycles are given followed by local radiotherapy, most often 2 Gy x 20. Six cycles are given for stage I bulky disease and/or elevated LDH and stage II1, otherwise 3 cycles. With elevated LDH, a sign of high rate of proliferation in the tumor, CHOP14 with rituximab may be used. In cases where 6 cycles are given and there is complete remmission after 3 and 6 cycles, radiotherapy may be avoided. It is possible to replace radiotherapy by giving more cycles (total 6-8) in situations where irradiation will cause high morbidity (for examplel to the oral cavity) or unwanted doses to organs at risk (for example kidneys or heart). A new study possibly indicates there is no benefit to giving radiation therapy after 4 CHOP cycles for patients over 65-70 years. Where radiation therapy is assumed to cause bothersome side effects, it is recommended to avoid radiation therapy after 4 cycles, especially in patients over 70 years.
When radiation therapy is given after limited chemotherapy mentioned above, CTV is defined as the original tumor volume with 2 cm margins in the craniocaudal direction. In the transversal plane, the original tumor volume is defined as CTV, but usually includes the full width of the entire region. If the original tumor volume has pushed aside healthy organs without infiltration and the organs have relocated to their original location after chemotherapy (lungs, kidney, intestines), it is correct to use the tumor's expansion in the transversal plane after chemotherapy for modeling of CTV (balloon effect). Margins for ITV will depend on the assumed internal movement of the area giving margins to field borders 3-4 cm in the cradiocaudal direction and 2-3 cm in the transversal direction.
Modeling of fields on the simulator will follow the same principle, that is, 3-4 cm from the macrotumor before chemotherapy to the field border in the craniocaudal direction and 2-3 cm in transversal direction.
Radiation therapy has a limited role for advanced stages (stages II2-IV). Some recommendations suggest that originally bulky lesions and extranodal manifestations should be irradiated after chemotherapy, but the benefit of this is unclear. This is not generally recommended. Localized residual lesions after adequate chemotherapy (6-8 CHOP + rituximab-based cycles) should be considered for biopsy and/or PET examination. Radiation therapy is considered consolidative. For positive biopsy of residual areas, younger patiens should be evaluated for 2nd line chemotherapy and consolidative therapy with HMAS. Residual tumor with 1 cm margin is defined as CTV, but the full width of the entire region is normally included. Margins for internal movement (ITV) are additional depending on localization. For direct modeling on a simulator, equivalent guidelines are followed. Bone involvement, if there are 1 or 2 bone manifestations, should normally be consolidated with radiotherapy. Original size in bone with 1 cm margin represents CTV. Original testicular involvement leading to irradiation of the scrotum with remaining testicles is done even after full chemotherapy with complete response.
Standard fractionation for DLBCL after full chemotherapy is 2 Gy x 20. For large volumes, there are limitations regarding organs at risk especially after HMAS where somewhat lower doses are considered, preferably 2 Gy x 16-18. Hyperfractionated treatment may also be considered with dose fractionation of 1.8 Gy with equivalent change in total dose if late toxicity from critical organs with high α/β ratio indicates that this is sensible.
Radiotherapy may be considered when starting primary treatment, especially with threatened organ function and pain caused by local growth. Radiotherapy can give good response and better quality of life even in lymphoma patients with advanced, chemo-resistant disease. If expected survival time is short, one should consider reduction of dose or fewer fractions with larger fractionations than normal.
Mediastinal (thymic) large cell B-cell lymphoma is a subentity of DLBCL that occurs in the mediastinum and has distinct clinical immunophenotypical and genetic characteristics. The clinical profile may be dominated by local aggressive growth with a tendency for vena cava superior syndrome and thrombosis of large veins in the mediastinum, the neck, and lower extremities. The biopsies may be dominated by fibrosis, and gene expression profiles of PMBCL have shown relationships between this entity and Hodgkin's lymphoma of nodular sclerosizing type.
Treatment follows guidelines as for DLBCL otherwise. There will usually be residual lesions in the medistinum even after 6 or 8 CHOP-rituximab cycles, and many patients must be considered for irradiation of these lesions. The role of radiation therapy in treatment of PMBCL is still uncertain, and since patients are often young, late toxicity in the form of cardiotoxicity and secondary cancer is significant. As a practical solution to this dilemma, radiation therapy should be avoided in patients in CR or CRu. Patients with PR but without sign of vital tumor (by biopsy or PET) are considered for radiation therapy (tumor-directed) after 6 cycles, when the cumulative anthracycline dose is still low at the time of radiotherapy. The total dose may be reduced to 32-36 Gy, and hyperfractionated treatment in fractions of 1.8 Gy is a possibility.
Treatment of Burkitt's lymphoma in adults 18-55 years follows the GM-ALL B-ALL/NHL 2002 protocol.
In this protocol, radiotherapy is recommended on residual tumor after 6 cycles (CRu and PR) with 36 Gy. Patients in CR will continue after 6 cycles, but initial mediastinal tumor or CNS involvement will be irradiated to the mediastinum to 36 Gy or total brain with 24 Gy. Patients in CR after 6 cycles, but initial extranodal involvement may be considered for consolidating radiotherapy to these areas, but this is not specified in the protocol. Patients with stage I or II without mediastinal tumor and without extranodal involvement which are in CR after 4 cycles should not have radiotherapy.
These patients are treated from ALL protocols where use of radiotherapy is often specified. For many patients, radiotherapy will not play a role in treatment. Radiotherapy is appropriate for CNS manifestations in combination with other CNS-directed treatment or as part of CNS prophylaxis for certain subgroups of patients. Radiotherapy for the testicles with testicular involvement is debated. Total body irradiation is also appropriate for some patients who are candidates for stem cell transplantation. There are retrospective data indicating the mediastinum should be irradiated with initial tumor involvement, probably also when CR is reached with chemotherapy. The dose should be upward to 30 - 36 Gy, but at Oslo University Hospital a maximum of 2 Gy x 15 after HMAS is used.
With irradiation of the mediastinum, it is often normal to define the intial tumor volume with a 1 cm margin in the craniocaudal direction. In the transveral plane, the residual tumor after chemotherapy is used as the basis (balloon effect), but the entire mediastinum/hilum is normally modeled in the CTV in the transversal plane. Margins are allowed for internal movement of the mediastinum. Modifications may be appropriate with tumor which has infiltrated for example the lungs or thoracic wall. Modifications may also be necessary to avoid too large a dose to organs at risk. It is often necessary to deviate from irradiation of the entire original craniocaudal mass and instead focus of the location of the residual tumor.
Aggressive T/NK-lymphomas (peripheral T-cell lymphomas without closer specification, anaplastic large cell T/0 cell lymphoma, etc.) rarely occur in localized stages (stage I/PeI – II1/PeII1). If this is the case, they may be treated according to guidelines analagous to confined stages of DLBCL. It may then be appropriate to irradiate to the involved field as for DLBCL. Standard fractionation is 2 Gy x 20.
For patients with advanced disease, radiation therapy to residual lesions is considered after adequate chemotherapy. Considerations are the same as for DLBCL.
Radiotherapy to the brain, especially with PCNS lymphomas enhances dementia. Newer protocols strive to avoid or postpone radiotherapy until possible recurrence. When radiotherapy is indicated, 2 Gy x 20 is given to the total brain field.
Oslo University Hospital shall not be liable for any loss whether direct, indirect, incidental or consequential, arising out of access to, use of, or reliance upon any of the content on this website. Oslo University Hospital© 2018


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Chris Eubank Jnr "I'm not old school like my father & I'm not new school like most other fighters, I'm my own school"
Chris Eubank Jnr on his Amateur career
Ringnews24.com: Could you tell us a bit about your amateur background?
Chris Eubank Jnr: 26 fights, 24 wins. I won the Nevada State Golden Gloves and Western Regional Golden Gloves in the US. In England I won the 2011 international Haringey box cup tournament
Why did you turn Pro now, with the Olympics in London only months away, was it not possible for you to make Team GB?
Chris Eubank Jnr: To get on the Olympic team you have to fight and train in England but for most of my amateur career I was in Las Vegas. It meant there was no guarantee I would have made the team so I did not want to risk losing out on the high quality training I was getting in the states to come back to England to pursue the uncertain Olympic route.
Chris Eubank Jnr on his Professional career
Ringnews24.com: On your debut you came out to Tina Turner's "Simply The Best", possibly the greatest entrance song of all time. Will you be keeping the song as your entrance music throughout your career?
Chris Eubank Jnr: I can't tell in the future but for the time being that will stay as my entrance song.
Ringnews24.com: You have now had two fights, do you get pre-fight nerves? and what do you do to help battle these nerves?
Chris Eubank Jnr: I don't get nervous… I may get a bit anxious waiting in the changing rooms wanting to get out there and perform, buts that's about it.
Ringnews24.com: You looked very impressive on your debut, how did you feel the fight went?
Chris Eubank Jnr: It went perfect, boxed well, got my combinations off and felt great.
Ringnews24.com: You learnt a lot with the Jason Ball fight, were you happy with you performance considering you fought it mostly one handed?
Chris Eubank Jnr: Mostly? I didn't throw a single right hand so the word you are looking for is 'completely' one handed. And, yes, I was very happy with the performance, beating a tough experienced opponent with only my left is something to be proud of and will never be forgotten throughout my career.
Ringnews24.com: You have now completed two fight camps, how do these camps compare to the amateur training camps? Do you have any favourite exercise or least favourite?
Chris Eubank Jnr: The camps are the same, but as I progress as a fighter I will increase the difficulty/intensity of the training as I get stronger… I always try to push myself to the limit. Sparring is my favourite exercise… everything else is secondary.
Ringnews24.com: Being on channel 5 is giving you great publicity, its an opportunity to get recognized so early on how do you feel about being in the spot light so early on in your career?
Chris Eubank Jnr: I'm happy to have so much exposure so early on in my career. It's a great thing for the fans to see me grow as a fighter and it's great that boxing is back on terrestrial TV where the general public are able to get a look at the sport without having to pay. Having all these eyes on me pushes me to train and fight harder.
Chris Eubank Jnr on his dad
Ringnews24.com: Chris, having such a famous fighter as a father do you think this will hamper your career, as fans may expect to much early in your fights?
Chris Eubank Jnr: It is an advantage having a famous father.. I know I have skills. I've learnt my craft and because I have a name on top of that I have the opportunity to showcase my fighting ability to millions of people… what more can a fighter ask for.
Ringnews24.com: Do you feel any pressure to achieve as much as your father did in the ring?
Chris Eubank Jnr: All I have to do is worry about myself, not about my father.. if I can do that (which I can) I will be fine.
Ringnews24.com: Your dad has a great knowledge in the game. It must be great always knowing hes available for advice, what advice has he given you so far?
Chris Eubank Jnr: Be your own man.
Ringnews24.com: Have you considered going to the USA like your father did, to gain his early experience in the fight game?
Chris Eubank Jnr: I will fight wherever I can to gain the most experience possible.
Ringnews24.com: Your fathers been involved in so many great fight's what is your favourite fight of his and why?
Chris Eubank Jnr: Even though it ended in tragedy, the Watson II was the most impressive. He showed more heart in that fight than most fighters do in their entire careers.
Chris Eubank Jnr on the Future
Ringnews24.com: What are your plans for 2012? How many fights do you think you can fit into the year?
Chris Eubank Jnr: I am looking to fight at least 5 more times this year.
Ringnews24.com: How fast do you hope to push for the Lonsdale belt and other titles? do you have a time frame in mind?
Chris Eubank Jnr: I take each fight as they come; I don't look past anyone. I will fight for a title when I am ready and the time is right.
Message for the fans
Ringnews24.com: Finally, do you have a message for your fans?
Chris Eubank Jnr: I'm not old school like my father and I'm not new school like most other fighters, I'm my own school… So get ready to attend.


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Paper Cuts Collection Edgers
Viewer Comments
These are beautiful edgers. I particularly like the sunflower. You gals are all so talented with what to do with everything. Love your ideas!
Love these edgers! Connie R's idea for the scrapbook page is also a good idea for making a bulletin board border (classroom, office or teachers lounge)
Love those edgers! Add special touch to cardmaking! Thanks for the ideas.
Thank you for some different ideas.
Beautiful dies and card ideas!
I am wondering if anyone has tried this type of die for creating 12" long borders for scrapbook pages (since I scrapbook much more than making cards)?
I think I will have to purchase one of the edger dies and try them for longer scrapbook borders.
Thanks, Connie R.
Reply from Paper Wishes Customer Service:
Hi Connie, I think the edgers would work really well with scrapbooking. You could cut one from the top edge of 12"x4" paper and then realign and cut again below the first one. Great idea!
Paper Wishes Weekly Webisodes feature free scrapbooking video classes and card making video classes! Every week Paulette and LeNae bring you new online scrapbooking or card making classes demonstrating the very latest paper craft and scrapbooking techniques. Each of our free card making and scrapbooking classes also gives you unique project ideas and tips from Paper Wishes' Design Team. Come back often so you don't miss any of the fun in our card making and scrapbooking classes! You are sure to be inspired! Also watch for our "Week of Webisodes" events where we feature new scrapbooking video classes daily!


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text: Ecommerce 80% Of Snapdeal Employees Will Be Terminated Soon; The Road Ahead is Tough! Mohul Ghosh Aug 1, 2017 0 Snapdeal has rejected the Flipkart deal, but it is going to directly affect their employees. The new pivot from Snapdeal is proving to be an axe to nearly 80% of Employees.


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Summer Lunch contributes to £3,000 Yorkshire Air Ambulance Donation
Sunday 22nd July was the Lions Summer Lunch at Settrington when over 100 Lions and guests enjoyed a wonderful Hog Roast prepared by Fryton Catering. The weather contributed to a very successful fund-raiser for the Yorkshire Air Ambulance at the start of President Steve's year.
President Steve and Anne encountered some interesting wildlife in the garden of John and Sue Harrison who hosted the lunch! The raffle was very well supported and Steve had a few words of thanks for all who attended.
When all costs are taken into account the event raised just short of £2,000 and this was made up to a £3,000 donation from our club funds.
Thank you to John and Sue Harrison for hosting the event, to all those who helped set up and take down the marquees and to everyone who attended and supported us.


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With Big E finally being cleared for in-ring competition, The New Day brought their best to the trio of Dolph Ziggler, Kevin Owens and Sami Zayn in the SmackDown LIVE main event. Elsewhere, Shane McMahon attempted to make an example out of The Miz as his Super ShowDown victory tour continued.
With the odds stacked against him, The Miz got the chance to get a piece of Shane McMahon tonight by way of defeating Elias and Drew McIntyre in singles bouts. The A-Lister was undeterred by the mountain he was facing, and with Shane and McIntyre lurking at ringside, he took down The Living Truth down with a Skull-Crushing Finale to get one step closer to colliding with Shane-O-Mac.
That was just the first step for Miz though, as The Scottish Psychopath entered the ring to prey on a worn-down A-Lister. Miz battled through and staggered the massive McIntyre at points, but the combination of what he went through against Elias, plus a distraction from Shane and McIntyre's sheer brutality led to Miz being finished off by a ring-rattling Claymore Kick for three.
Despite Miz not being victorious, Shane-O-Mac took advantage of the situation by calling for a match against the down and dazed A-Lister. Shane-O-Mac's plan almost backfired though when Miz fired back on Shane, but Miz's exhaustion was too much to overcome, with Shane getting the better of him and then placing Miz in his signature Triangle Hold for a "win" before another excessive celebration.
With her newfound friend of Alexa Bliss in her corner, Nikki Cross looked to tear through SmackDown Women's Champion Bayley in one-on-one competition.
The Goddess has been filling Nikki's head with allegations that The Hugger isn't as sweet as she seems, telling Nikki that Bayley liked several disparaging tweets about her last night. With Cross seeking payback for the supposed tweets against Alexa's WWE Stomping Grounds opponent, WWE's Twisted Sister was clearly fired up as she stepped to the champ. Her eagerness paid dividends at times, with Bayley not always having an answer for her aggressive attack, but The Hugger persevered and put Cross down with a massive elbow drop for the win, making a major statement to Bliss.
WWE Champion Kofi Kingston looked to yet again vanquish the three Superstars that have been coming for his head since he captured the title but this time with his New Day brothers by his side, as Kingston, Xavier Woods & Big E clashed against Dolph Ziggler, Kevin Owens & Sami Zayn in the blue brand's main event.
In the clutch, Ziggler accidentally nailed Zayn with a superkick, which allowed Kingston to strike with two Trouble in Paradises – one to The Showoff, which took him out of commission, and then another to Zayn, which secured the victory for The New Day.
The New Day was back, and yup, they clearly still rocked.
In other action … The Miz and Shane McMahon nearly came to blows on "Miz TV" … Heavy Machinery def. local competitors … Sonya Deville def. Carmella … and Sonya Deville def. Carmella.


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Implementation Guidelines
WISE adopts the following implementation guidelines in its programme activities.
Focus on economic independence and working against free hand-outs and dependency syndrome. Identifying own assets (strengths, opportunities, resources, etc.) and building on them. Bringing attitudinal changes among the low-income community and partners for achieving transformed lives.
Involving target groups, their spouses, families, and the community in the programme. Diversifying target groups by including low-income women with disabilities, women with HIV/AIDS, illiterate women, women-headed households, migrant workers, returnees, and street beggars.
Learning and sharing
Continually learning from own experience as well as others and sharing good practices and information with a wider public so that the programme will have spill-over effects.
Ensuring the sustainability of the programme through capacity building of the institutions, cost sharing, leadership development, income generation, and SACCO membership with the Union.
Collaborating and networking with different organizations and participating in networks and forums to promote learning and replication of WISE's programme as well as advancing implementation of action plan and excellence.
Addressing, to the extent possible, the critical issues of development of the target group to ensure holistic development.
Continual improvement
Security, safety and health
Ensuring, to the extent possible, the security of employment of the target members. Promoting the concept of safety and health in one's occupation both for staff and target group. Ensuring the protection of target members and their children in connection with their relationship with staff or visitors.
Reflecting regularly on performance to learn from experience and enhance innovation and continual improvement in the quality of services provided to the target group and partners. Excelling in performance in all regards.


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Red-Pink Baby Leaves Inner Tube Earrings
Regular price £18.00 Sale price £14.00
Sexy and lovely handpainted inner tube leaves made out of a scrap bicycle inner tube. The large leaf has a silver, and the little leaf has a red colour.
Surprise people around you with your new upcycled earrings. You will never find another handmade jewellery like this.
Full length: 3.25"
Fitted on a nickel free hook.


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redim as pcg32 b(1000000) will create one million generators!!!
Each generator is asked for one random number and then an average is determined.
It is no bloody wonder the code takes a long time. Each new generator created goes past MyRandomize which uses FB's #5.
Above I show how to use PCG32II.
I will definitely write a Help file.
If the intention is to determine the average of 1000000 random numbers in [0,1) got from ONE generator then this is how it is done.
Code: Select all
#Include Once "PCG32II.bas"
Dim As Ulong i
Dim As Double tot
Dim As pcg32 pcg
For i = 1 To 1000000
tot += pcg.randse
Print tot/1000000


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Our municipal attorneys are experienced in the formation of special purpose entities and districts, including fire protection, water and sewer, public utilities, irrigation, cemetery, transit and public facilities districts. We counsel our municipal clients during all phases of forming a special purpose district. We guide our clients through initial hearings to determine need and feasibility, the election process if needed, and the establishment of governance for newly formed special districts. In addition, our land use attorneys can assist during the SEPA review process to which all proposed special districts are subject.
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