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Cell division occurs during normal human development and aging. Despite the likely importance of cell division to human pathology, it has been difficult to infer somatic cell mitotic ages (total numbers of divisions since the zygote) because direct counting of lifetime numbers of divisions is currently impractical. Here we attempt to infer relative mitotic ages with a molecular clock hypothesis. Somatic genomes may record their mitotic ages because greater numbers of replication errors should accumulate after greater numbers of divisions. Mitotic ages will vary between cell types if they divide at different times and rates.
Age-related increases in DNA methylation at specific CpG sites (termed "epigenetic molecular clocks") have been previously observed in mitotic human epithelium like the intestines and endometrium. These CpG rich sequences or "tags" start unmethylated and potentially changes in methylation during development and aging represent replication errors. To help distinguish between mitotic versus time-associated changes, DNA methylation tag patterns at 8–20 CpGs within three different genes, two on autosomes and one on the X-chromosome were measured by bisulfite sequencing from heart, brain, kidney and liver of autopsies from 21 individuals of different ages.
Levels of DNA methylation were significantly greater in adult compared to fetal or newborn tissues for two of the three examined tags. Consistent with the relative absence of cell division in these adult tissues, there were no significant increases in tag methylation after infancy.
Many somatic methylation changes at certain CpG rich regions or tags appear to represent replication errors because this methylation increases with chronological age in mitotic epithelium but not in non-mitotic organs. Tag methylation accumulates differently in different tissues, consistent with their expected genealogies and mitotic ages. Although further studies are necessary, these results suggest numbers of divisions and ancestry are at least partially recorded by epigenetic replication errors within somatic cell genomes.
Human "age" can be characterized by chronological age (time since the zygote) and mitotic age (total number of divisions since the zygote). Somatic cells within an individual have similar chronological ages but their mitotic ages may differ because different cell types divide at different rates and times. Cell division occurs during normal human development and aging, and many diseases are associated with excessive numbers of divisions. Despite the likely importance of cell division to human pathology, it has been difficult to measure mitotic ages because human lifetimes are long and direct observations or experimental manipulations are impractical.
Here we attempt to infer relative somatic cell mitotic ages with a molecular clock approach [1]. Divisions should be surreptitiously recorded within a genome because the greater the number of divisions, the greater the number of replication errors. Molecular clock approaches are commonly employed to study species evolution and infer ancestral trees. All life is thought to be related to a universal ancestor that existed billions of years ago [2]. Sequences differ between individuals and species because genomes cannot be duplicated exactly, and replication errors accumulate. Similarly, all cells within an individual are related, share an ancestral genome, and eventually trace their ancestry back to a universal common ancestor called the zygote (Figure 1). Somatic replication errors are likely to accumulate as genomes are copied during development and aging. Sequence comparisons could be used to infer the mitotic ages of human cells, but sequences mutate too infrequently during a lifetime to effectively function as somatic molecular clocks. For example, point mutations are rare (< 1 per 100,000 bases) even in cancer cell lines [3]. With this error frequency, one would have to sequence millions of bases to identify a few somatic replication errors in normal tissues. Microsatellite loci mutate at higher frequencies and various investigators have proposed or provided data illustrating their potential to reconstruct somatic cell ancestry [4,5].
Figure 1. A human somatic cell tree. A: Every cell has a genealogy that starts with the zygote and ends with its present day phenotype. The genealogy of a differentiated cell can be divided into three phenotypic phases – development from the zygote, a stem cell phase, and differentiation. B: Stem cells are common ancestors in a somatic cell tree. The zygote is the ultimate common ancestor, and adult stem cells are more recent common ancestors of differentiated cells. C: Replication errors record cell division or genome duplication. Illustrated are a 5' to 3' series of eight CpG sites that are initially unmethylated (open circles). Random replication errors may accumulate during cell division such that some CpG sites become methylated (filled circles). The greater the numbers of divisions since the zygote (mitotic age), the greater the number of errors or methylation (molecular clock hypothesis).
It may be possible to replace the 5' to 3' order of bases with the 5' to 3' order of CpG DNA methylation because genome replication also involves the duplication of epigenetic patterns. Methylation also exhibits somatic inheritance [6], but is a binary (methylated or unmethylated) code. Unlike sequences, methylation measurably changes with age in certain mitotic human tissues at specific CpG sites [7], suggesting epigenetic replication error rates may be greater than genetic replication error rates.
DNA methylation patterns may be directly compared between different aged individuals because methylation is removed early in development (before implantation) at most CpG islands [8,9]. This early active and passive demethylation essentially synchronizes epigenomes between individuals and potentially an age-related increase in subsequent methylation at certain CpG rich regions ("epigenetic molecular clocks") may represent an accumulation of epigenetic replication errors. Bisulfite sequencing studies of mitotically active epithelium like human endometrium, colon, and small intestines reveal age-related methylation consistent with a "clock-like" accumulation of stochastic replication errors [10-13].
Replication errors may represent the major source of age-related methylation because genomes are regularly duplicated in mitotic tissues. In addition, cell division appears to be required for in vitro de novo methylation [14]. Potentially the mitotic history of any cell can be reconstructed from its methylation patterns if such patterns are not "created" but instead represent a lifelong accumulation of replication errors. Cells with greater mitotic ages would generally have more errors or methylation relative to cells with fewer divisions. However, some methylation may occur independent of division, which would confound a somatic cell clock analysis. To further test whether methylation at "epigenetic molecular clocks" occurs independent of division, brain, heart, kidney, and liver tissues from individuals of different ages were examined. Cell division is minimal in adult brain and heart cells [15,16], and age-related methylation should be absent in these tissues if the majority of tag methylation represents replication errors. Adult liver and kidney cells normally divide infrequently but can divide in response to damage [17,18].
Specimens were obtained from 21 autopsies performed at the Los Angeles County-University of Southern California Medical Center (Table 1). The ethical use of the human tissues for research was approved by our Institutional Review Board. Intervals between death and tissue collection were generally less than three days. Heart specimens from 18 individuals were obtained from grossly normal right or left ventricle. Brain specimens from 18 individuals were obtained from grossly normal frontal cortex (primarily gray matter) or cerebellum. Liver specimens from 12 individuals were grossly normal except for the cirrhotic livers from Patients M and Q. Kidney specimens from 18 individuals were obtained from the cortex and appeared grossly normal.
Table 1. Specimens
DNA was purified from approximately 25 to 50 mg of tissue using a column method (Dneasy, Qiagen, Valencia, CA). Approximately 500 ng of DNA was bisulfite treated for 12 hours at 50 C using an agarose bead method [11]. Bead sizes were about 25 to 30 ul.
Methylation analysis
Approximately 10% of the agarose bead containing bisulfite treated DNA was amplified for each PCR (42 cycles) of three different CpG rich loci or tags (Figure 2 and Table 2) that previously exhibited age-related methylation in epithelial tissue [11,19]. The first tag is in the 3' transcribed but untranslated region of the CSX gene (also called NKX2-5). This 51 bp tag contains 8 CpG sites and exhibits age related methylation in epithelial tissues such as the colon, small intestines, and endometrium [11-13]. The second tag is a 151 bp region in exon 2 of SOX10 that contains 20 CpG sites. The SOX10 tag appears to be a relatively fast somatic cell molecular clock because it becomes nearly fully methylated in hair by two years of age [19]. Finally, a 77 bp region in exon 1 of BGN just 3' to the promoter that contains 9 CpG sites and exhibits age related methylation in the colon [11] was examined in the heart and brain of males. The BGN tag is on the X-chromosome, and there is only a single allele per cell in males. PCR products were visualized on an agarose gel and cloned (TOPO TA Cloning kit, Invitrogen, Carlsbad, CA). Individual bacterial clones were sequenced to obtain seven or eight tags per locus per specimen. Clones with evidence of incomplete bisulfite conversion (C's at non-CpG sites) were discarded from the analysis.
Figure 2. Epigenetic molecular clocks or tags. A: Sequences (after bisulfite conversion) of the CSX (8 CpG sites), BGN (9 CpG sites), and SOX10 (20 CpG sites) tags. Primer sites are underlined, CpG sites are in bold, and a bisulfite converted "C" at a non-CpG site is indicated by "T". B: Examples of CSX and SOX10 tags sampled from the heart, brain, liver or kidney of different aged individuals. The tags are arranged in a 5' to 3' horizontal order with methylated sites indicated by filled circles. There are eight alleles per tissue. Patient H did not have a liver specimen and the liver is instead from Patient G, a 31 year old.
Table 2. Expression of tag loci*
Percent methylation is calculated by dividing numbers of methylated CpG sites by total numbers of examined CpG sites. Statistical analysis used linear regression to test for age-related changes in methylation, and a two-sided t-test for differences between specimen groups. Significance was set at p < 0.05.
The specimens examined in this study are described in Table 1. Methylation was examined at three CpG rich regions or tags (Figure 2 and Table 2). These tags were initially chosen because they exhibit age-related methylation in epithelial tissues and their genes are not expressed at high levels in epithelium. However, some of the tags are expressed at higher levels in the tissues examined in this study (Table 2). CSX is highly expressed in the heart [20] and BGN has higher expression in the brain and heart. Expression may prevent methylation regardless of cell division because promoter methylation is associated with gene silencing [21]. Of note, only the BGN tag is located close to its promoter in exon 1.
Mitotic age and stochastic replication errors
Mitotic age cannot be directly inferred from tag methylation because of the random or stochastic nature of replication errors. For example, methylation replication error rates for the CSX tag have been estimated at about 10-5 to 10-4 per CpG site per division [11]. Therefore, many cells will lack tag methylation even though they have divided many times. Cells with tag methylation may have either fewer, the same, or greater numbers of divisions than cells with less tag methylation.
The inability to link methylation levels directly with numbers of cell divisions is illustrated by simulation for CSX and SOX10 tags (Figure 3). For a given mitotic age, individual tag methylation patterns and values are scattered. However, average values of eight tags are less scattered. Therefore, the experimental studies are based on average measurements of seven or eight tags per specimen. Consistent with stochastic errors, tissue methylation patterns were diverse (Figure 2).
Figure 3. Simulations of random methylation errors at the CSX and SOX10 tags. For CSX tags, the simulated error rates are 1 × 10-4 per CpG site per division for methylation and 2 × 10-5 for demethylation. SOX10 tags appear to become methylated faster in human tissues, and the simulated error rate for methylation is greater (1 × 10-3) with the same demethylation error rate as CSX. Errors are assumed to be independent between CpG sites, and each tag is an independent genome (no shared ancestry). These simulations are presented to illustrate the general stochastic nature of mutation, and are not an attempt to model the experimental data. A: Frequencies of individual tag methylation values and averages of 8 tags after 500 (green), 1,000 (red), 2,000 (blue), or 5,000 (gray) simulated divisions. As expected, values are more scattered for individual versus average tag values. B: Examples of simulated individual CSX or SOX10 tags after different numbers of divisions.
The heart continues to grow in size after birth, at rates comparable to body weight [22]. CSX and SOX10 tag methylation was significantly lower in fetal, infant and children (≤ 5 years old) hearts and greater in adult (> 21 years old) hearts (Figure 4 and Table 3). Age-related changes in CSX or SOX10 methylation were not observed in adult hearts. BGN heart tag methylation also did not exhibit age-related changes and was generally low (< 5%), with no significant differences between adult and young (≤ 5 years old) hearts. There were no significant differences in tag methylation between the right and left heart.
Figure 4. Heart tag methylation. A: Tag methylation with chronological aging. Circles are averages of eight tags with red solid dots from the right heart, and blue X's from the left heart. Trend lines (black, all values; red, right heart; blue, left heart) were not significantly different from a level line (linear regression, p values > 0.05). B: Histograms of individual tags comparing individuals before heart growth is largely finished (≤ 5 years of age, gray bars, N = 64, 63, and 37 alleles for CSX, SOX10, and BGN tags) and adults (black bars, N = 161, 143, and 94 alleles for CSX, SOX10, and BGN tags). CSX or SOX10 tag methylation values were significantly higher in adults.
Table 3. Heart and brain tag methylation
Brain growth and development occurs early in development and is essentially complete by two years of age [15]. Statistically significant age-related changes in CSX, SOX10, or BGN tag methylation were not observed (Figure 5) after infancy (> 2 years of age). Significantly lower levels of CSX and SOX10 tag methylation were found in neonatal brains (Figure 5B and Table 3). CSX and SOX10 tag methylation levels were significantly greater in the cerebellum compared to the cerebral cortex (Figure 5C and Table 3). However, there was no significant difference in BGN tag methylation between the cerebellum and cortex, or between neonatal and adult brains, but overall tag methylation levels were low (< 10%).
Figure 5. Brain tag methylation. A: Tag methylation with chronological aging. Circles are averages of eight tags with red dots from the cerebellum and blue X's from the cerebral cortex. Trend lines (black, all values; red, cerebellum; blue, cortex) were not significant different from a level line (linear regression, p values > 0.05). B: Histograms of individual tags comparing individuals before brain development is largely finished (< 2 years of age, gray bars, N = 72, 72, and 56 alleles for CSX, SOX10, and BGN tags) and older individuals > 2 years of age (black bars, N = 144, 144, and 88 alleles for CSX, SOX10, and BGN tags). CSX or SOX10 tag methylation values were significantly higher in older individuals. C: Histograms of individual tags comparing methylation between the cerebellum (gray bars, N = 40, 40, and 24 alleles for CSX, SOX10, and BGN tags) and the cortex (black bars, N = 104, 104, and 64 alleles for CSX, SOX10, and BGN tags) from older individuals (> 2 years of age). CSX and SOX10 tags were significantly more methylated in the cerebellum. Note that there is a high frequency of completely unmethylated SOX10 tags in the older cortex, which may reflect the presence of neural cells that express SOX10 and therefore preclude methylation (see Discussion).
Liver and kidney
CSX and SOX10 kidney or liver tag methylation levels were significantly higher in adults relative to individuals less than 21 years of age (Figure 6 and Table 4). There was an age-related increase in tag methylation with adult kidneys or livers, but these trends were not statistically different from a level line. BGN tags were not examined in the kidney or liver.
Figure 6. Liver and kidney methylation. A: Tag methylation with chronological aging in the liver. The trend line indicates an age-related increase in methylation, but the data were not significantly different from a level line (p = 0.38 for CSX, p = 0.19 for SOX10, linear regression). Red solid dots indicate the two samples with cirrhosis. Tag methylation was significantly greater in adults (black bars, N = 80 and 72 alleles for CSX and SOX10 tags) compared to younger individuals ≤ 5 years of age (gray bars, N = 32 alleles for CSX and SOX10 tags). B: Tag methylation with chronological aging in the kidney. The trend line indicates an age-related increase in methylation, but the data were not significantly different from a level line (p = 0.59 for CSX, p = 0.097 for SOX10). Red solid dots indicate samples with benign nephrosclerosis. Tag methylation was significantly greater in adults (black bars, N = 105 and 104 alleles for CSX and SOX10 tags) compared to younger individuals ≤ 5 years of age (gray bars, N = 40 alleles for CSX and SOX10 tags).
Table 4. Kidney and liver tag methylation
Cirrhosis was present in two livers, one of which had the highest SOX10 tag methylation and high CSX tag methylation (Pt M). The other cirrhotic liver (Pt Q) had high CSX tag methylation but not remarkably high SOX10 methylation. The liver with the highest CSX tag methylation was from a patient with AIDS (Pt L), with no obvious liver pathology. The two kidneys with high CSX tag methylation had benign nephrosclerosis, which was not observed in kidneys from individuals of similar age with lower CSX methylation. Average CSX and SOX10 tag methylation was higher in kidneys with benign nephrosclerosis, but the average age of individuals with benign nephrosclerosis was also higher, consistent with the observation that nephrosclerosis is more common in the elderly [23].
Tag changes in multiple tissues
Trend lines for average CSX or SOX10 tag methylation with age for heart, brain, liver and kidney are illustrated in Figure 7A. For comparison with prior studies [11-13,19], average CSX and SOX10 tag methylation with age are also illustrated for colon, small intestine, endometrium and hair in Figure 7B. In general, tag methylation is low around birth and higher in adults. Statistically significant age-related increases in CSX tag methylation were observed in colon, small intestines, and endometrium. SOX10 tags become methylated faster than CSX tags and age-related changes are not observed because they are nearly fully methylated in adult tissues, especially in mitotic epithelium.
Figure 7. Average tag methylation with chronological age for multiple human tissues. A: Trend lines for brain (blue), heart (red), liver (brown), and kidney (purple). Negative ages represent fetal tissues (birth equal zero years). B: Trend lines for colon (black, data from ref. 11 and additional unpublished specimens), small intestines (black dotted, data from ref. 12), endometrium (gray dotted, data from ref. 13), and hair follicles (gray, data from ref. 19). SOX10 tag values for intestines and endometrium are unpublished data from three different aged individuals (six glands and 48 tags from each organ).
Although much is known about human development and aging, the exact fates of somatic cells are somewhat mysterious because it is difficult to observe cells throughout a lifetime. Potentially somatic cell mitotic ages may be inferred by counting numbers of replication errors using a molecular clock approach [10]. Here we provide further evidence that somatic genomes likely record divisions as they are duplicated and physically passed from generation to generation. Certain methylation patterns at specific CpG rich sequences or tags appear to represent somatic replication errors and therefore surreptitiously record mitotic ages.
Prior studies of mitotic adult tissues (intestines and endometrium) demonstrated age-related tag methylation consistent with methylation representing replication errors [11-13]. Cell division is initially required for normal heart and brain development but is infrequent in adulthood [15,16]. Consistent with tag methylation representing replication errors, adult brain and heart tags were methylated but failed to demonstrate age-related increases. The adult tag methylation likely represents replication errors acquired much earlier in life during development because methylation was lower in fetal and infant tissues. Therefore, tag methylation patterns appear to be stable for decades in the absence of division.
Cell division occurs rarely in normal adult liver, perhaps once a year [17]. Consistent with low division rates, tag methylation increased with chronological age, but this trend was not significantly different from a level line. Relatively higher tag methylation levels were observed in livers with cirrhosis, consistent with observations that cirrhosis is associated with hepatocyte proliferation [24]. Division in normal kidney is thought to occur but is relatively rare [18]. Consistent with low mitotic activity, tag methylation apparently increased with age but this trend was not statistically significant. Relatively higher tag methylation was observed in kidneys with benign nephrosclerosis, a relatively common finding at autopsy that increases with age [23].
Comparisons between different human tissues
A molecular clock approach can compare genomes in cells of widely different phenotype. For example, sequences potentially infer ancestry across all three kingdoms of life [2]. Similarly, a somatic cell clock analysis may allow relative comparisons between cell types if their replication error rates are similar. CSX and SOX10 tag methylation with chronological age for multiple tissues are illustrated in Figure 7. Tag methylation was lower early in life for all tissues, consistent with a start with unmethylated tags and a common origin for their genomes. Methylation or errors increased early in life, consistent with the hyperplasia needed for growth and differentiation. Division largely ceases after birth for the brain or heart, and their mitotic ages and numbers of replication errors appear to remain fixed for a lifetime. In contrast to the heart, brain, liver and kidney, there were statistically significant age-related increases in methylation in the epithelial tissues [11-13]. Generally, endometrium from older women appears to have the highest average mitotic ages, with relatively lower mitotic ages in the heart and brain.
Different tags may have different replication error rates and attain different levels of methylation. Tags with higher error rates better document early development because the mitotic ages of neonatal and infant tissues are relatively low. For example, SOX10 tags become nearly fully methylated by infancy and therefore only minimal changes are possible even with further increases in mitotic age. By contrast, tags such as CSX with lower error rates exhibit fewer changes early in life but more readily document further mitotic age changes during aging.
Human somatic cell genealogy
Mitotic age contains additional information about somatic cell genealogy because only certain cell types divide during the differentiation from a zygote to present day cells. Somatic cell genealogy can be divided into three successive phenotypic phases – development from the zygote, a stem cell phase, and differentiation (Figure 1). Genealogy may be confined to certain patterns because numbers of divisions during development and differentiation may be restricted to characteristic numbers and times. We have observed methylation patterns consistent with three basic human genealogies (Figure 8).
Figure 8. Types of observed human somatic cell genealogies. A: Static genealogy observed in adult non-mitotic tissues (heart, brain, kidney, liver). Genealogical phases are indicated by green (development from the zygote to the tissue stem cell), red (stem cell phase), and blue (differentiation). Adult genealogies and mitotic ages are fixed with respect to chronological age and replication errors in present day cells accumulated much earlier in life. B: Continuous genealogy observed in mitotic epithelium. Only the stem cell phase can vary with chronological age because numbers of divisions are relatively constant during development and differentiation. The mitotic age of a differentiated cell is the mitotic age of its stem cell plus the few additional divisions required for differentiation. An age-related increase in replication errors and mitotic age depends on stem cell divisions. C: Punctuated genealogy observed in hair follicles. Average mitotic age and replication errors do not increase with age despite the high hair follicle mitotic activity because hairs and their follicles are cyclically lost and destroyed every few years. Bulge stem cells initiate each new hair cycle, and the mitotic age of a differentiated follicle cell is the age of the stem cell that initiates the new cycle plus the divisions in the follicle. New hairs would have essentially the same mitotic age regardless of chronological age because bulge stem cells are relatively quiescent and only divide a few times at the start of each new hair cycle [26], or if there is clonal succession [25] in which new hair cycles are initiated by previously latent stem cells.
The first pattern, observed in non-mitotic adult tissues such as the brain and heart, is a static genealogy. Mitotic age remains constant during chronological aging, and replication errors found in adult cells primarily represent divisions during earlier organ developmental and differentiation. Liver and kidney also have this general pattern, but further stem or differentiated cell divisions may occur during adult life from normal cell turnover or in response to cell loss.
A second pattern, observed in mitotic epithelium such as the intestines and endometrium, is a continuous genealogy that expands as replication errors accumulate throughout life. Replication errors likely accumulate primarily in adult stem cells because stem cell lineages are long lived and numbers of divisions during development and differentiation are usually similar regardless of chronological age. For example, intestinal development is limited to the neonatal period and differentiated cells in the intestines divide only a few times and survive about a week [25]. Therefore, only the stem cell phase can have variable numbers of divisions with aging. The mitotic age of a differentiated intestinal cell is the mitotic age of its stem cells plus the few divisions required for differentiation. Stem cells in the colon, small intestines, and endometrium appear to divide throughout life because their differentiated cells contain greater numbers of replication errors or methylation with increasing chronological age.
A third pattern, observed in hair follicles, is a punctuated genealogy. There is a lack of a measurable age-related increase in average replication errors despite high hair follicle mitotic activity [19]. This pattern may occur if stem cells exhibit mitotic latency, either by dividing infrequently or through clonal succession [26]. Human hair grows for several years and then is lost when the differentiated cells in the mitotic compartment or bulb physically disappears. A new follicle and hair regrow after several months, initiated by stem cells that reside in the hair bulge [27]. Bulge hair stem cells are normally quiescent and divide primarily at the start of a new hair cycle. This stem cell quiescence (infrequent division) or clonal succession (latent stem cells successively divide) ensures that the average mitotic age of hair bulb cells does not measurably increase with chronological age because each new hair cycle starts from a "young" stem cell. Mitotic age is reset when differentiated but mitotic bulb cells (and their errors) are eliminated at the end of a hair cycle, and the next new hair has essentially the same genealogy regardless of chronological age. Physical configuration may influence genealogy, with a punctuated genealogy observed with episodic follicle destruction and reformation, and continuous genealogies observed with glands that persist throughout life.
Epigenetic clocks and selection
Selection can alter error frequencies by favoring or eliminating some types of errors. Methylation can potentially confer selection because promoter methylation is associated with loss of expression, and promoter methylation is another mechanism to silence tumor suppressor genes in cancer [21]. The loci studied (CSX, SOX10, BGN) were initially chosen because these genes were not highly expressed in epithelium and therefore any methylation would likely be neutral. However, some of these genes are expressed at higher levels in the heart and brain (Table 2), and evidence that expression prevents methylation was observed for the BGN tag, with relatively little methylation (< 10%) even in adult brain or heart. By contrast, CSX is highly expressed in the heart [20], but CSX tags appeared to record mitotic age with less methylation early in life and stable (~18%) adult heart methylation. The CSX tag is in the 3' untranslated region of its gene, which may not affect or be affected by its transcription, whereas the BGN tag is just 3' to the promoter in exon 1. The SOX10 gene is expressed in only some neural tissues, mainly those of neural crest origin [28]. Fully unmethylated SOX10 tags were rarely found after birth in any tissue, except in the cerebral cortex (Figure 5), suggesting SOX10 expression in certain neural cells may preclude or prevent methylation in the region (exon 2) of our tag. Although the mechanism responsible for methylation at our tags is uncertain, methylation does appear to be reduced when the tag is located near an active promoter.
Replication errors are stochastic and the methylation of a single allele is relatively uninformative. Our analysis sampled a relatively small number of tags from only a few different loci, but illustrates the potential for "somatic cell clocks" to record mitotic ages. More information can be obtained with more tags from the same "clock" locus or from multiple "clock" loci. Methylation may not be independent between CpG sites and error rates may vary between CpG sites within a tag or between cell types. However, with most replication error mechanisms, average methylation should generally correlate with mitotic age. Another biological source for tag variation is the sampling of heterogeneous cell types. In this study, organ fragments containing uncertain mixtures of different cell types were analyzed. Ideally tags from small homogeneous clones of cells (as with our prior epithelial studies of single glands [11-13]) would be measured because each cell type may have a distinct genealogy.
The present study provides tentative evidence that somatic cell mitotic ages may be recorded within their genomes by methylation changes at certain CpG rich loci. The exact mechanisms responsible for methylation changes at our "epigenetic molecular clocks" are uncertain, but many changes appear to represent replication errors. Mitotic age in actively dividing adult tissues may largely depend on the behavior of their stem cells because numbers of divisions during development and differentiation are limited. There are a number of challenges to inferring genealogies from replication errors, but such an approach potentially allows for systematic studies of human development and aging without prior experimental interventions.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MWC, CLE, and JYK performed most of the experiments and helped write the manuscript. ASY and GCK supplied specimens. KDS and ST helped with the statistical and quantitative analysis. DS analysed the data and wrote the manuscript.
This work was supported by a grant from the National Institutes of Health (CA111940). ST is a Royal Society-Wolfson Research Merit Award holder and is supported in part by a grant from Cancer Research UK.
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• A hands on approach to solving problems
• Available to work
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Selling a signed brand new 2014 St George Illawarra NRL jersey.
Call Larry - 0412654255
plumber free gas blockage waterheater
We are looking for two housemates for our Marrickville sharehouse.
Located on Horton St, we are walking distance to Marrickville, Sydenham, Addison and Illawarra roads. Two seconds from Addison rd markets, the Henson, 15 min walk to Sydenham train station, 5 min bike ride to Enmore rd. It's a quie...t, sunny street that backs onto Henson oval.
The house is large with a large backyard and a currently empty car spot.
We have two bedrooms that need to be filled by July 26.
One bedroom is s
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Smallish room available in Marrickville flat above shop. 4x3m (big enough for queen bed, desk, dresser etc then some), large 3 bedroom flat living with 2 guys (30 & 34) working full time. 2 mins from Marrickville station on Illawarra rd. surrounded by shops, cafés and some really good restaurants pl...us across the road from woolies. Wifi included. Bond is 4 weeks rent plus 2 weeks rent in advance. $170 pw. Would suit someone who can live w 2 musicians who works and doesn't mind a drink.
Hit me ...
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Motorcycle Parts Superstore Visit: ebay or www.f1moto.com.au
Urban Garden And Tree Care
Walk for dollars, deliver catalogues get paid to exercise anybody can deliver. Tues/Wed and Sat/Sun on an independent contracting basis you choose your hours.
Great for adults, students, families or groups, including retirees, pensioners and migrants.
Flexible hours.
Close to home.
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Car is good but some areas may not need one just depends.
Telephone and Email is needed.
Honest, reliable people wanted who would like to earn some extra money in the
Please respond with
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I am searching for a bedroom for a nepalese couple in illawarra area. Please let me know if anyone is looking for couple to share.
Affordable professional photography

subset: commoncrawl


text:
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Showing 17 to 24 of 50 posts
Due to the bad weather my trip to Fontainebleau in Februrary left quite a bitter taste, but fortunately March offered another opportunity to visit the marvellous forest south of Paris. Thats how I happened to enjoy three weeks of Fontainebleau with a motivated group from Innsbruck. It wouldn't be Font however, if the weather wouldn't show itself to be moody, with hail, rain and snow (again), which cut the climbing days down to 12. I wanted to do a lot, and my ticklist was really long – of course way too long for Bleau :-)
Still I managed to climb some very beatiful problems, like "Appartenance 7c", "Dosage 7c", "Noir desir 7c", "Supplement d'armes (stand 7b+ and assis 7c+)" and "Nemesis 7a". Additionally I made good progress in some projects, and so the motivation to return is huge!
Following a short video of the trip with some problems like "La rampe" 7a; "Supplement d´armes" 7b+ ; "Manolo" 7c; "Verdict" 8a und "Noir desir" 7c.
 Read More...
In the past view weeks spring has finally arrived here in Austria, the cold and snow has vanished of our valley grounds, uncovering the near by blocs in the Ötztal from their hibernation... I personally love winter for all it's beauty and pleasures it offers here in the center of the Alps, hiking up the mountains and ride powder really can catch up with climbing that's for sure! But at the same time I can't wait to escape the crowded and dusty gyms and go climb for real again as soon as the local crags are on.
April has been a really busy month for me - but in a good way! I've contested in different national and international competitions in all but one weekend of this month. All competitions were bouldering comps.
On the 6th of April I brought my strongest junior girls near Copenhagen to a city called Lyngby to contest in this year's first round of the danish bouldering competition called Bloc Comp.
The competition consisted of a qualifying round and of course the finals. With twelve out of thirteen possible tops, most of them flashes, I was in the lead position for the finals.
On the first problem in the final round I made a huge mistake. Not just one of the sloppiness alike "oh why did I just crimp that hold when it's much better as a pinch?" climbing related or technical mistakes, but one of those big, big fails that one simply can't do during a bouldering final. Completely distracted by whatever, I went out to the first problem in the final. I placed both hRead More...
Here is a small piece of an amazing steep roof located in Southern Utah, hopefully it will cheer everyone up after the burglary, all the best and a big thanks goes out to Ben Moon for always making the coolest clothes and keeping us all psyched to do our best!
Read More...
The winter didn't start in the best of the ways this year. Maybe because of personal engagements, that kept me in my home area...
Maybe because of the terrible weather conditions we have had the last three months in Italy... Lets call them a series of unfortunate circumstances. :)
Winter Part 1 Video
Imagine you´re scrambling through the undergrowth for a while than you turn around a big boulder which seems to be like any other one (and there are thousands) and there it is! What looks like just another high quality boulder, which is not that untypical for this area, turns straightforward into one of my favorite boulder problems of all time. Basically the whole setting of the problem is just overwhelming. The boulder is surrounded by high mountains with snow on there peaks and lies between many other rocks which are thrown together in a narrow valley. Nobody would expect such a perfect landing in this chaotic block field, but it is like that. It´s sand like in Font! At first sight you think it´s a smaller boulder problem until you start to check out the moves and now you realize that it´s bigger than it looks. The holds are perfect and exactly at the right position.
It´s a phenomenon that´s not only with this problem, but for me on Heritage it´s obvious. If there would be Read More...
Just after Christmas Holiday, I focused my "climbing time" on bouldering because the temperatures were too cold for sport climbing. I decided to visit some good bouldering spots with my brother and some friends.
The first place we visited was Donnas, in Aosta Valley where we and a lot of people are discovering and brushing lots of good new problems. I had two boulders in my mind Klettermachine 8a and Mucca Pazza 7c+. I started trying Klettermachine because I did the "Stand start" in fact I had to link just the first one move to complete the full boulder. After 30 minutes of work I climbed all the moves and I was super satisfated! Klettermachine is done, It's Mucca pazza time! :D
I had some problems understanding the moves of Mucca pazza, because it's really powerfull boulder composed of five moves on bad slopers and funny toe/hell hooks, COMPLETLY MY ANTI-STYLE! I needed two days of hard work to figure out all the moves and then I CrRead More...
And the video!
From dirt grows flowers / 8cAnd the video!
From dirt grows flowers / 8cRead More...


text: Hard to believe, but Carhartt has been making the working man's life better for 125 years now. Celebrate this remarkable feat with this Made-in-the-USA acrylic watch hat. It's made of stretchable, 100% acrylic rib-knit fabric for warmth and comfort. The Carhartt 125th anniversary logo is sewn on the front of this classic, one one-size-fits-all hat.


text:
Settle Down Jesus Christmas Mug
(approx. $7.73 or €7.01)
- Product Type: Mug
- In Stock
- Usually Ships in 1-2 Days
- UK & Worldwide Delivery
- StockID: 160696
Add a splash of humour to your Christmas meal with the help of this hilarious mug! As you sip away on your mulled wine, this cheeky mug featuring the captions 'Settle Down Jesus' and 'Hey, Fuck You! It's My Birthday!', is sure to add to your Christmas cheer!


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In This Section
Journal Forum
Audibility of a CD-Standard A/DA/A Loop Inserted into High-Resolution Audio Playback - September 2007
13 comments
Sound Board: Food for Thought, Aesthetics in Orchestra Recording - April 2015
2 comments
Reflecting on Reflections - June 2014
4 comments
AES Journal Forum
Auditory Evacuation Beacons
[Engineering Reports] Auditory evacuation beacons can be used to guide people to safe exits, even when vision is totally obscured by smoke. Conventional beacons make use of modulated noise signals. Controlled evacuation experiments show that such signals require explicit instructions and are often misunderstood. A new signal was designed that combines a chime sound with a spoken message ("exit here"). In a tunnel environment the evacuation success rate without prior instructions to the participants was 16% for conventional beacons and 87% for the newly designed beacons. Also, a novel way for coding the relative distance to the exit was used. By exploiting the precedence effect through the application of time delays, subjects were induced to naturally "follow" the sound. During an experiment in the mockup of a ship's interior 88% of participants followed the intended route compared to 38% with the conventional approach. The optimum time delay between beacons was found to be approximately 20 ms.
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DIY wall storage bins. All this project takes is some old crates and pretty paint, and your foyer, front hall, or mud room will look cute AND organized. #Brother
52 Brilliant and Smart Kids Rooms Storage Ideas (47) #storagesolutions #craft #diy #kids #happykids #happy #shoes #storage www.larkandcastle.com
Related Pins
apple crate bookshelf <- why didnt i think of this, considering my daddy works in produce?! totally love!
OH GOODNESS...I love love love this shoe storage or whatever storage. It is off the ground & easier to reach & clean under (also leaves space for super tall rain or work boots if needed)...awesome idea!
I love stand alone storage units for coats, backpacks and purses! I could use one at every door in our house.
We are doing something like this on our landing. Bead board floor to ceiling with a shelf up high, hooks, bench bottom with shoe storage underneath. It's in the plans for this summer!!!
I bet I could do this on a larger scale with zip ties for a cool stack structure on a tabletop for a craft show display!
Great idea... but only if you don't have children. madame-lala.blogs...


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36 year old man | Cedar Park, TX, USA | Seeking women 28-40 within 40 miles
Active within 5 days
Well let's see here I grew up in a small town...
Well let's see here I grew up in a small town in south Texas by Corpus Christi. My parents raised me to respect my elders, open doors for ladies, use my manners and to be a gentleman. Get along with just about everyone, like meeting new people, and my Mama thinks I'm pretty awesome. I have two daughters. They are beautiful and they are the most important part of my life. They bring me joy, so you better like kids! I do enjoy cooking for family and friends! My fried fish and shrimp is to die for… my grandfathers old recipe. I also like to coach youth baseball; right now I am coaching an 8U baseball team at CPYL. Many of the parents don't understand why I'm out there if I don't have a son playing but the kicker for me is that I love the game. So what do I like to do for fun……I like to do things outdoors when I can, I like to go fishing, camping, and I would like to say I'm good at most sports. Not cocky just good. I am a huge baseball and football fan and I am always up for attending a game. I love me some college football…Texas Longhorns. I'm looking for someone who is a little country and a little city, someone who doesn't take life too seriously. Someone who is sweet, caring, has a good sense of humor and can be my partner in crime.
Camping , Cooking , Fishing/Hunting , Gardening/Landscaping , Exploring new areas , Playing cards , Playing sports , Travel/Sightseeing , Volunteering , Watching sports
Baseball, Football, Golf, Running, Skiing, Tennis / Racquet sports
I exercise 1-2 times per week.
I like Cats, Dogs, Horses
I don't have any animals right now but if I had any animal it would be a Blue Heeler. We have a pet Hamster "Lucy"!
Middle of the Road
Texas A&M University–Kingsville, Kingsville, TX
I love to watch the Texas Longhorns in person or on TV. I also love playing with my daughters! They make the world go around for me!
I like to play softball on my free time or coach one of my kids sports team. I like going to the lake and hanging out. Enjoy going to watch the TEXAS LONGHORNS (any sport but mainly football and baseball)
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What he is looking for
|5'10" (177cms)
|5'0" (152cms) to 5'10" (177cms)
|Body type:
|About average
|Slender, About average, Athletic and toned, A few extra pounds
|No preference
|Light brown
|Auburn / Red, Light brown, Dark brown, Blonde, Dark blonde
|No Way
|No Way, Yes, but trying to quit
|Never, Social Drinker
|Executive / Management
|No preference
HSE Manager
|$50,001 to $75,000
|No preference
|No preference
|Have kids:
|Yes, and they sometimes live at home (2)
|No preference
|Wants kids:
|Not sure
|No preference
|White / Caucasian
|Latino / Hispanic, White / Caucasian
|Christian / Other
|Christian / Catholic, Christian / Protestant, Spiritual but not religious, Christian / Other
|Some college
|No preference


text:
Product Specs
Product Description
Dress tables in style with these elegant glitter bow place cards. Available in your choice of shimmery gold or silver, these fancy place cards will add dazzle to tables or buffet bars. Just add your personal note for a pretty, easy, and functional birthday party accessory.


text:
Harvick holds off Busch for NNS win at Atlanta
Aug 31, 2013, 10:32 PM EDT
Kevin Harvick led 132 of 195 laps, but had to hold off a hard-charging Kyle Busch in order to come away with a NASCAR Nationwide Series victory tonight at Atlanta Motor Speedway.
Harvick had kept the lead following pit stops under yellow at Lap 186 and lined up for the subsequent restart alongside Joey Logano. Logano spun his tires, allowing Harvick and Busch to break away and settle the race themselves.
With six laps left, Busch got a good run on the outside of Harvick through Turns 1 and 2 to take a half-length lead on Harvick down the backstretch. But Harvick held firm on the inside and was able to come back and clear Busch off Turn 4 to complete what would be the race-winning pass.
"We did a great job on pit road," Harvick said to ESPN in Victory Lane. "We were definitely too tight, and I had to change my approach. I knew Kyle was gonna go for it.
"Three years in a row, we've had the dominant car [in this race] and you know, you think something's gonna go wrong. But we just kept digging and these guys did a great job all weekend."
Busch finished half a second behind Harvick, with NNS points leader Sam Hornish Jr. in third, Kasey Kahne in fourth, and Kyle Larson in fifth. Logano was unable to recover after his poor final restart, and settled for sixth.
In the NNS championship, Hornish increased his lead over Austin Dillon to 10 points after the latter finished eighth in tonight's race.
- chugginbud - Sep 1, 2013 at 9:22 AM
Harvick will regret leaving RCR!
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- NHRA: New president Peter Clifford full of ideas to get drag racing back on-track 10
- Ryan Briscoe making most of filling in for James Hinchcliffe 1


text:
Lancome Cils Booster XL
427 reviews
73% would repurchase
Package Quality: 4.1
Price: $$$
Package Quality: 4.1
Price: $$$
INGREDIENTSAqua/Water/Eau, Oryza Sativa Cera/Rice Bran Wax, Cera Carnauba/Carnauba Wax/Cire De Carnauba, Cera Alba/Beeswax/Cire D'abeille, Stearic Acid, Candelilla Cera/Candelilla Wax/Cire De Candelilla, Triethanolamine, Palmitic Acid, Acacia Senegal/Acacia Senegal Gum, Panthenol, Sodium Polymethacrylate, Hydroxyethylcellulose, Propylparaben, PEG/PPG-17/18 Dimethicone, Methylparaben, Simethicone, Rayon, Polyquaternium-10, Tocopheryl Acetate, Polycaprolactone.
on 8/5/2010 11:03:00 PM
Age: 25-29
Skin: Acne-prone, Fair-Medium, Warm
Hair: Brunette, Wavy, Medium
Eyes: Brown
I'm gonna have to say, this primer did nothing for my lashes. I could not even tell a difference. My lashes did not stay curled, it did not lengthen or thicken and they were super clumpy(clumpy is not thick in my eyes). I usually like clumpy but this was too much for me. I used the entire tube, but will not buy it again.
on 8/1/2010 3:49:00 AM
Age: 25-29
Skin: Combination, Fair, Not Sure
Hair: Brown, Other, Other
Eyes: Brown
I am pissed at this mascara base and pissed off at myself for giving in and spend $42.50 to buy the freaken Lush lash set from Nordstrom. I heard a lot of good reviews on it so i decided to buy it without trying it first. Stupid, i know. The freaken cilis booster xl mascara base sucked and did nothing for my lashes. In fact it weighed down my lashes, even after i curled my lashes with an eye lash curler. After applying the mascara base, and seeing that my lashes have fallen back into their normal state, which was straight downward pointing, i didn't even want to apply the mascara anymore. And when i did apply the Hypnose mascara, I wanted my money back. Sooooo bad. Even when i applied the mascara without the base, i was still disappointed. I just wish i had read more reviews before i bought it. My advice is that if you have very straight stubborn lashes and most mascaras don't give your lashes a lift, you WILL be disappointed if you buy this, because it will make your lashes droopy. I warned you. Now, how am i ever going to finish up the freaken mascara base and the hypnose mascara? So mad.
on 7/29/2010 7:36:00 PM
Age: 19-24
Skin: Very Oily, Fair, Not Sure
Hair: Brown, Wavy, Medium
Eyes: Brown
I haven't had a really noticeable different when using this primer. It also doesn't hold my curl at all, so by the time I'm ready to apply my mascara over this, my lashes have already straightened out again. It also clumps very easily.
on 7/17/2010 9:25:00 PM
Age: 30-35
Skin: Dry, Olive, Not Sure
Hair: Black, Other, Other
Eyes: Brown
Of two minds about this product. have used with both diorshow blackout and hypnose.
CONS: straightens my lashes dead straight after curling with a mascara wand. makes the lashes clumpy and agree with others that it looks a bit spidery.
PROS: first product ever (!!) that does not cause my mascara to give me raccoon eyes and believe you me i've tried every mascara under the sun. maybe it's always because i have always needed a primer. does make my lashes look a bit fuller and longer.
will not be buying again as i have very straight lashes and love to have that curled look, but will be using the rest of the tube/wand as it was $27...
definitely recommend testing this product first before paying for it.
on 7/7/2010 4:19:00 AM
Age: 18 & Under
Skin: Acne-prone, Medium, Not Sure
Hair: Brunette, Straight, Medium
Eyes: Brown
I have been using this mascara base for 4 years now and I love it. It is my HG mascara base. I've tried Shiseido's The Mascara Base and I did not like it at all. This is a bit pricey but totally worth it for boosting whatever drugstore mascara you're layering over it. This product looks white on the wand but it's actually invisible when applying it. Must be some kind of magic... Anyway, it really lengthens your lashes and gives volume. This makes any crappy mascara look great.... well maybe not any... but most.
on 6/19/2010 10:42:00 PM
Age: 18 & Under
Skin: Combination, Fair-Medium, Not Sure
Hair: Black, Straight, Medium
Eyes: Black
This products irritated my eyes plus it drooped my eyelashes...def will not buy again....waste of money. lancome mascaras overall are just overhyped....and arent that good
on 6/17/2010 7:35:00 PM
Age: 19-24
Skin: Combination, Medium, Not Sure
Hair: Black, Other, Other
Eyes: Brown
This is nearly neck to neck with my Smashbox primer, which is my HG of all mascara primers. It works well and helps give clean definition and length. A little more pricey, but it is worth it.
on 6/5/2010 10:24:00 AM
Age: 25-29
Skin: Sensitive, Fair-Medium, Neutral
Hair: Brunette, Wavy, Medium
Eyes: Brown
I LOOOOOOOOVE Lancome mascaras....................but the price tag is just too ridiculous. I use mascara and primer every day, and replace every 3 months, so its just not realistic for me to spend $30 bucks a tube on an ongoing basis. That being said, this primer has it all...great formula, great brush, makes any mascara look like falsies.
on 6/4/2010 2:48:00 PM
Age: 36-43
Skin: Combination, Olive, Not Sure
Hair: Brunette, Other, Other
Eyes: Brown
Used the sample and love it so far! Makes my lashes super long and thick when paired with my favorite Lancome mascaras (Courbe and Definicils). Tried even with a Clinique mascara sample and looks fab.
on 5/21/2010 4:21:00 PM
Age: 19-24
Skin: Combination, Dark, Warm
Hair: Black, Kinky, Coarse
Eyes: Brown
please understand this is great, I honestly feel people who say it is not must either be onto a really good primer that costs like 50 quidies or not using it properly. It is a creamy white primer and when applied it should be coated over the lashes completely.I find the key is not to apply the mascara straight away or it will just take off some of the primer on your mascara brush so you do not get the full effect. However, you must also not let it dry or it will be clump-o-rama and make lashes feel brittle and mascara will not apply evenly. What I do is apply primer on 1 eye then the other, then quickly zig zag the base returning to the first eye and then do the second. There is a noticeable difference when I use this, my lashes are so long that once I went to Oxford street london and no lie, everyone kept stopping me to tell me I had beautiful eyes. Once I ran out of this and I made my boyfriend run to a department store (I live in Knightsbrige, London...PHEEWWW) to get me 1 as I refused to go out. This is how much this primer means to me, it is a necessity. I am not even lying when I say I have dreams and even in my dreams I am wearing this.
Popular Mascaras


text:
Seibertron.com - The Ultimate Transformers Fansite
Blurrz wrote:Although the first wave of Beast Hunters Deluxes all featured retooled First Edition or Robots In Disguise figures
, the stock picture provided makes it unclear whether or not the First Edition mold is retooled for Beast Hunters Starscream.
Va'al wrote:I keep track of everyone. Backwards.
There are atandarfs to maintain.
LOST Cybertronian wrote:Hey, If Mindmaster survived then you should do just fine.
william-james88 wrote:So that would also be the case for Predaking, who is also six members.
njb902 wrote:Why oh why do we keep getting stupid weapons.
Rated X wrote:This website is starting to become just like the NBA. (US Basketball) When you lightly brush up against someone's shoulder, the referee calls a foul. When you disagree with the referee's call, he threatens to eject you from the game.
Rated X wrote:Beast Hunters seems to be destined to fail. The whole "beast" concept wasnt liked by everyone back in the 1990's. I think bringing it back will slow down the momentum of the TF franchise.
Return to Transformers Toys Discussion
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text:
Forty-five sets that capture the weekend's mud, madness, and mischief.
Released Albums
Unreleased Albums
Upcoming Festival Appearances
Berlin, Germany (Tempelhof Airport)
Sep 12 - Sep 13 2015
Updated 1 month ago
Biddinghuizen, Amsterdam
Aug 21 - Aug 23 2015
Updated 2 months ago
Hasselt, Belgium (Kempische Steenweg)
Aug 20 - Aug 22 2015
Updated 2 months ago
Austin, Texas (Zilker Park)
Oct 02 - Oct 11 2015
Updated 2 months ago
Byron Bay, Australia (North Byron Parklands)
Jul 24 - Jul 26 2015
Updated 3 months ago
Chicago, Illinois (Grant Park)
Jul 31 - Aug 02 2015
Updated 3 months ago
San Francisco, California (Golden Gate Park)
Aug 07 - Aug 09 2015
Updated 3 months ago
Wiltshire, England (Larmer Tree Gardens)
Sep 04 - Sep 06 2015
Updated 4 months ago
Isle of Wight, England (Robin Hill)
Sep 10 - Sep 13 2015
Updated 5 months ago
Governors Ball 2015 Festival Review: From Worst to Best
on June 09, 2015, 11:15am
In Photos: Bunbury Music Festival 2015
See what kept the Queen City in jubilation this weekend
on June 08, 2015, 9:00pm
Top 10 Music Festivals and Headliners in North America: Spring 2015 Power Rankings
To quote Bob Dylan, "Things have changed."
on June 05, 2015, 6:35pm
Puppet versions of Tame Impala star in "'Cause I'm a Man" video — watch
The Dominic Simper puppet is pretty spot on.
by Ben Kaye
on May 29, 2015, 12:45am
Sasquatch! 2015 Festival Review: From Worst to Best
A rising star tops the Gorge's fantastic four-day weekend.
by Dusty Henry
on May 27, 2015, 9:15am
Boston Calling Spring 2015 Festival Review: From Worst to Best
Beck, My Morning Jacket, and St. Vincent keep the city's namesake festival strong.
by Ben Kaye and Edward Dunbar
on May 25, 2015, 4:00pm
Tame Impala share dizzying video for "'Cause I'm A Man" — watch
A continually spinning 3D model of a relationship in motion.
by Ben Kaye
on May 21, 2015, 10:40am
Lollapalooza reveals 2015 schedule: Here are the 10 worst conflicts
Paul McCartney, Flying Lotus, or The Weeknd? alt-J or First Aid Kit? The War on Drugs or Alabama Shakes?
by Alex Young
on May 19, 2015, 11:05am
Shaky Knees 2015 Festival Review: From Worst to Best
It was a sizzling three-day weekend in Hotlanta.
on May 11, 2015, 1:15pm
Tame Impala premiere new song "Eventually" — listen
Another glowing track from the band's forthcoming album, Currents.
on May 07, 2015, 3:45pm
Update: Austin City Limits announces 2015 lineup
Foo Fighters, The Strokes, Drake, Florence and the Machine, and many more set to descend on Austin, TX this October.
by CoS Staff
on May 04, 2015, 12:07am
Release date and tracklist revealed for Tame Impala's new album Currents
Psych rockers' forthcoming LP spans 51 minutes and features 13 tracks.
on May 01, 2015, 5:23pm


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text: The World's End Trailer by Justin Dweeber at May 8, 2013 11:06 am. Updated at August 22, 2013 10:29 am. Comments Share Tweet Email The first trailer for The World's End. The sci-fi comedy closes out Simon Pegg and Edgar Wright's Blood and Ice Cream Trilogy. Edit Delete Follow Movie Trailers? Tag: Movie Trailers Related Videos: Movie Trailers Related Posts: The World's End Trailer: Watch Now! The World's End Reviews: How Does the Cornetto Trilogy Wrap Up? Uploaded by: Justin Dweeber Uploaded: May 08, 2013 Modified: August 22, 2013 10:29 Duration: 152 seconds


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No Doubt Tribute No Duh With Vitalogy and Red Not Chili Peppers
* Additional fees apply. No coupon or promo codes necessary to enjoy the displayed discount price.
The last date listed for No Doubt Tribute Band No Duh With Vitalogy and Red Not Chili Peppers was Friday November 18, 2011 / 7:30pm.
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Rock Icons The Smashing Pumpkins and Marilyn Manson Celebrate The End Times Tour
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We are rockers and big pearl jam fans, so went for RNCP and Vitalogy. RNCP singer and band were pretty good, better than expected, and impressed. Vitalogy was excellent and portrayed early PJ very well, even through they played a couple songs that were not early in PJ's career (OK by us). The singer and band were having fun and were well received. We had wanted to see them before but kept having conflicts went they played locally. After this performance we will always see them when they play local and don't have a conflict. After two better than expected performaces, we stayed to check out No Duh but after 3 songs left. They were over the top and just not very good in our opinion but again we are not into that type of music.
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Great way to go out on a discounted price. we had 7 people attend this event and had a great time


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Cards bullpen blows it... Again. Up 3-0 after 7, lose 4-3 in 10.
"Man, the pitch looks fast, even in slow motion." Thom Brennaman on Chapman's fastball.
What did make me happy, however, was seeing the Cubs balk in the tying run and Posey putting them ahead.
I'm bringing this oldie out just because:
It's Patriots Day, 11AM start for Rays-Red Sox if anyone's able to watch or listen.
"Reality tells us there are no guarantees. Except that some day Jon Lester will be on that list of 100-game winners." - Peter Gammons
Is today Jackie Robinson day?
Yep, 42, and taxes, just in case anyone's really been sleepwalking.
"Reality tells us there are no guarantees. Except that some day Jon Lester will be on that list of 100-game winners." - Peter Gammons
Take Julio Teheran out of the equation and the Braves' pitching staff has an ERA of 1.19. Incredible.
Heads of state that ride and wrangle. That look at your face from more than one angle. Can cut you from their bloated budgets. Like sharpened knives through Chicken McNuggets.
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Tampa Bay Lightning
Juniors Game Night: 12-27-14Submitted by chad on December 28, 2014 - 11:08
Western Hockey League
Calgary Hitmen 2 @ Kootenay Ice 6
D Ben Thomas, CAL: 0-0-0, -4, 0 PIM
Season totals: 25 GP, 3-11-13, E, 12 PIM
2013-14 totals: 72 GP, 7-24-31, +11, 39 PIM
Thomas returned after missing a few weeks with a lower-body injury. He was a team-worst minus-4 as Calgary outshot Kootenay, 53-26, but lost, 6-2. Wyatt Hoflin (undrafted) made 51 saves for the Ice.
Notes: The following players remain out of their respective lineups:
Brayden Point (Moose Jaw) is with Team Canada for the World Junior Championships
Anthony DeAngelo (Sarnia) is with with Team USA
Dominik Masin (Peterborough) is with Team Czech Republic
NHL Game Night: 12-27-14 Hurricanes at LightningSubmitted by pete on December 27, 2014 - 22:01
As dominant a 2-1 win as you can get.
Ben Bishop allowed 1 goal on 26 shots for the victory in his return from injury. He didn't have to make many key saves, but when they came he got them.
First Period
8:50 TB Kucherov (15), (Palat)
Second Period
Third Period
4:53 TB Killorn (8), (Callahan, Filppula)
14:04 CAR Sekera (2), (E. Staal, Gerbe)
Ondrej Palat, Alex Killorn, and Ryan Callahan were the game's three stars. I'll throw a shout out to Mark Barberio in there, who played his best all-around game since he was AHL Defenseman of the Year for the Norfolk Admirals.
If the Lightning had capitalized on their PP opportunities (0-for-6) the game wouldn't have been close. As it was, the chances were there but they were snake bitten. So, that's at least a sign things are progressing in the right direction.
Cedric Paquette had 1 hit and 1 blocked shot in 12:42. He was also 20% on draws. He kept up reasonably well playing with Stamkos and Connolly.
Box score and extended statistics from NHL.com.
NHL Game Night: 12-23-14 Penguins at LightningSubmitted by pete on December 23, 2014 - 22:35
Christmas done Triplets style.
Andrei Vasilevskiy allowed 3 goals on 29 shots for the victory. Tonight was light work until Third Period garbage time, which was a welcome relief after the volume of shots/chances he faced on Long Island.
First Period
6:54 TB Johnson (11), (Kucherov, Palat)
19:56 TB Johnson (12), (Palat, Hedman)
Second Period
0:52 TB Kucherov (14), (Carle, Filppula)
2:37 TB Johnson (13), (Kucherov, Palat)
10:15 PIT Hornqvist (13), (Klinkhammer)
Third Period
9:55 PIT Kunitz (10), (Malkin)
18:29 PIT Letang (8), (Hornqvist, Malkin)
Tyler Johnson, Nikita Kucherov, and Ondrej Palat were the game's three stars. Like Jon Cooper's top line reindeer bringing a sleigh full of winning.
This was a very important win for a number of reasons. One, it showed the thing that happened on Long Island was an aberration. Two, it allows the team to feel good headed into the Winter Break and they can start after the break with a pretty fresh slate. Third, it helped them bury the long losing streak they've had against Pittsburgh, which is a team they'll likely see over the next few years as a playoff adversary. True, the Pens were depleted. True the Pens were tired. I'm not under any delusions that the Lightning bested the Penguins' most fearsome lineup tonight. But, it's something positive to build on, and they did have Crosby, Malkin, and (for a little while) Fleury on the rink.
When the Lightning come back from break, the first 5 things they need to work on are: power play, power play, power play, power play, and power play. It's bad right now. Absolutely terrible. Time for the coaching staff to take some egg nog down into Cooper's laboratory and white board something fresh, because what they're doing right now is an atrocity masquerading as a travesty.
With 4 games to go in this 10-game segment, the Lightning sit 7 points shy of the 12 they would want per segment, but just 2 points shy of the spot they need to be at the end of the 4th segment (48 points) thanks to the insurance points they've squirreled away. 7 out of 8 possible points is tough, but you'd like to see the Lightning get at least 5 or 6 out the 8 possible points so they don't burn off too much of what they have in the bank in case another rainy day pops up.
Cedric Paquette had 1 shot and 2 hits in 13:29. I thought he played his role really well tonight.
Jonathan Drouin was -2 with 1 shot and 1 hit in 13:04. I'm not a huge fan of the Drouin/Stamkos/Connolly combination that was put together by Cooper tonight, because I'm not sure I see a lot of puck retrieval on that line unless Stammer does it.
Box score and extended statistics from NHL.com.
AHL Game Night: 12-20-14 Devils at CrunchSubmitted by pete on December 20, 2014 - 21:54
Fans went to a fight and another Syracuse Crunch win broke out.
Kristers Gudlevskis allowed 2 goals on 28 shots for the victory. That's 4 straight quality starts and 5 straight wins and he's run his save percentage up to .907 now.
First Period
SYR Angelidis, (11) (Marchessault, Namestnikov), 8:41 (PP)
SYR Blunden, (5) (Angelidis), 13:34
Second Period
ALB Boucher, (7) (Whitney, Scarlett), 7:32 (PP)
SYR Namestnikov, (4) (Nesterov, Samson), 9:30 (PP)
Third Period
ALB Thompson, (13) (Black), 7:56
Mike Angelidis, Vladislav Namestnikov, and Gudlevskis were the game's three stars. The Crunch ran their winning streak to 9 games as they continue to diligently stalk Manchester for first place in the Eastern Conference, where they remain just a point out. Given the Lightning's struggles up top, though, I would not be shocked to see Namestnikov moved back up sooner rather than later to try to shake the chemistry up in Tampa. Either way, though, the Crunch defense and goaltending seem to have it rolling now. They'll reconvene Friday against the Devils when I suspect there might be a roster change or three, because a defenseman may be going up, too.
Box score from TheAHL.com.
NHL Game Night: 12-20-14 Lightning at IslandersSubmitted by pete on December 20, 2014 - 21:46
Sluggish Lightning nearly bailed out by Vasilevskiy.
Andrei Vasilevskiy was brilliant. He stopped 45 shots tonight, only allowing a goal to an uncovered Tavares in the slot (no shame) and a tipped point shot by Lee (again, no shame). The Lightning would've lost this game 8-1, easily, without Vasilevskiy.
First Period
Second Period
Third Period
9:16 TB Connolly (6), (Johnson, Barberio)
16:51 NYI Tavares (13), (Okposo, Bailey)
17:03 NYI Lee (7), (Hamonic, Nelson)
18:49 NYI Kulemin (6), (Clutterbuck)(EN)
Vasilevskiy was the game's third star.
Anyone in a Lightning jersey not named Vasilevskiy or Boyle probably didn't deserve to be wearing it the way they played tonight. I certainly hope there's a more innocuous explanation for what we witnessed tonight. Maybe the flu is running through this team, which was aggravated by Garrison leaving in the Second Period. I can't explain it, otherwise. The team looked slow. They made wave after wave of really idiotic turnovers. They looked physically and mentally fatigued to the extent that they didn't even seem to want to be there. Gudas and Carle looked like corpses out there, for crying out loud. Getting outshot 48-21 by anyone is just not acceptable, and in the absence of extenuating circumstances there needs to be a closed door meeting followed by a bag skate, because this just cannot happen.
Cedric Paquette was -1 with 2 penalty minutes 2 hits and 1 blocked shot in 15:11. If Yzerman looks to make a shake up move, I wouldn't be shocked to see Paquette and Namestnikov trade places soon. Paquette took a bad offensive zone boarding call and he's seen his offensive contributions vanish while taking about 1 minor penalty a night. That means, sadly for him, he might have to be the one who gets shook in the shake up.
Jonathan Drouin had 1 hit in 13:19. He ended up getting himself pulled off the Stamkos line because they were generating nothing. Zero shots on goal and only one even attempted isn't good enough.
Box score and extended statistics from NHL.com.
NHL Game Night: 12-19-14 Lightning at DevilsSubmitted by pete on December 19, 2014 - 22:09
Lightning manage to wrestle 1 point out of the quicksand.
Evgeny Nabokov allowed 2 goals on 18 shots before allowing 1 of 3 to convert in the penalty shot session for the SO loss. I'll give him full marks for battling tonight, but that was hard to watch. The first goal was a fluky goal that he put into his own cage. Okay. But, the second goal cannot be allowed at any level of hockey and he got the benefit of 5 or 6 posts tonight on top of that. The Lightning have a real issue there, and the solution may be figuring out some way to shuttle one of the two good goaltenders they have in Syracuse up in back-to-back situations from here on out, which is a roster challenge, but may be necessary. You could've timed that second goal from shot to net with a sun dial it fluttered so slowly.
First Period
3:01 NJ Henrique (9), (Greene, Gomez)(PP)
15:17 NJ Elias (4), (Havlat, Henrique)
Second Period
14:55 TB Stamkos (19), (Callahan, Drouin)
Third Period
19:19 TB Kucherov (13), (Callahan, Garrison)
NJ- Elias
Nikita Kucherov was the game's first star.
Glass half full: they managed to squeeze a point out of a game where they trailed New Jersey in the Third Period. That's no small feat, and part of climbing out of the big hole that they were in was one of the better passing displays we've seen in the last 3-4 weeks, especially in the Second Period. I also thought Radko Gudas, coming off a stomach bug, might've played his best game of the season tonight at both ends of the rink.
Glass half empty: the goaltending was bad. The power play might've actually been worse, though. 0-for-5 on the power play tonight and one was a key double minor in the Third Period while they were pushing to tie the game where they got nothing going. Nothing. At some point as the season progresses, Yzerman seriously needs to find a righty PP QB to acquire via trade. Nothing they're doing to try to free up Stamkos with lefties at center point is working, and it's becoming counterproductive trying to force set pieces his way or asking him to force passes back through the seam to Filppula, who can't do anything with them because he's not a shooter.
Cedric Paquette had 2 hits and 1 blocked shot in 12:39 and he also was 44% on draws.
Jonathan Drouin had a helper and was +1 with 2 shots and 1 hit in 21:15. Circle that ice time number. Playing with Stamkos for the full game, this was the first time Cooper really let Drouin out of the stable, and I think Stamkos in particular responded well with 10 shot attempts tonight. My big thing with Drouin now, though, is that team's are playing him as a pass first guy. He needs to shoot it more. 2 shots is more than he's been getting, but he only had 3 shot attempts total. I want to see him at a minimum of 4 shot attempts a game from here on out. Stamkos won't mind, Jonathan. 91 likes rebound goals, too.
Box score and extended statistics from NHL.com.
NHL Game Night: 12-16-14 Lightning at FlyersSubmitted by pete on December 16, 2014 - 21:49
Vasilevskiy plays the role of the slump slayer.
Andrei Vasilevskiy allowed 1 goal on 24 shots for the victory in his first NHL start.
First Period
19:19 PHI Simmonds (14), (Streit, Giroux)(PP)
Second Period
1:50 TB Stamkos (18), (Drouin, Callahan)
6:34 TB Johnson (10), (Callahan, Kucherov)
Third Period
19:18 TB Filppula (6), (Paquette, Johnson)(EN)
Ryan Callahan and Vasilevskiy were the game's first and third stars. The night offered Lightning fans a brief slice of the future, with Vasilevskiy in net and Jonathan Drouin playing on the top line with Stamkos. Both had important hands in the outcome of the game, with a cameo appearance by young Cedric Paquette on the EN goal to seal the game.
Paquette had a helper and was +1 with 2 shots, 1 hit, and 1 blocked shot. He was also 67% on draws.
Drouin had a helper and was +1 with 2 penalty minutes and 1 blocked shot in 17:28. If I have a critique of Drouin, it's that I'd like to see him look to shoot a little more and generate some offense that way, too. I hope he stays on that line with Stamkos from here on out.
Box score and extended statistics from NHL.com.
NHL Game Night: 12-15-14 Lightning at PenguinsSubmitted by pete on December 15, 2014 - 21:52
Bad news in Pittsburgh.
Ben Bishop allowed 1 goal on 5 shots before leaving the game after the First Intermission with what was deemed a "lower body injury". Evgeny Nabokov allowed 3 goals on 21 shots the rest of the way for the loss.
First Period
19:09 PIT Sutter (7), (Scuderi)(SH)
19:33 TB Drouin (2), (Johnson, Kucherov)(PP)
Second Period
2:55 PIT Rust (1), (Ebbett)
19:26 PIT Downie (5), (Hornqvist, Malkin)(PP)
Third Period
7:47 PIT Dumoulin (1), (Downie, Spaling)
16:56 TB Kucherov (12), (Filppula)
The Lightning came out flying in the First Period, and were betrayed by a mixture of bad luck and great goaltending by Fleury. The one goal Bishop allowed was a shot that was going wide that took a goofy bounce off the knob of his stick and bounded into his net. It was so goofy and unlucky, I wouldn't be surprised if Bishop hurt himself kicking something in the locker room about his buzzard's luck. It was that kind of night. You then had Nabby handing Pittsburgh a free goal on a puckhandling gaffe and then a fourth goal coming off a deflection off Hedman. Meanwhile, the Lightning couldn't buy another goal on Fleury until a breakaway goal from Kucherov late in garbage time. Just nothing is going right now, and you hope that Bishop being held out for precautionary reasons turns out to be over something minor like a stubbed toe rather than something being broken or torn.
Cedric Paquette had 1 shot and 4 hits and was 60% on draws in 14:10.
Jonathan Drouin had a goal and 2 shots and 1 blocked shot in 14:50. He was probably one of the three best players on the rink for the Lightning tonight, and that was reflected in his promotion to the Stamkos line in the Third Period. He could've easily had a 3 point night tonight if the Lightning were so cursed right at the moment.
Box score and extended statistics from NHL.com.
NHL Game Night: 12-13-14 Lightning at CapitalsSubmitted by pete on December 13, 2014 - 22:03
Little things.
Ben Bishop allowed 3 goals on 27 shots for the loss.
First Period
Second Period
10:33 WSH Backstrom (6), (Wilson, Schmidt)
18:40 WSH Backstrom (7), (Ovechkin, Wilson)
Third Period
2:17 WSH Backstrom (8), (Ovechkin)
15:36 TB Johnson (9), (Carle, Kucherov)
17:29 TB Carle (3), (Stamkos, Garrison)
19:21 WSH Fehr (6), (unassisted)(EN)
Frustrating. This was a game filled with self-inflicted wounds. I'm not going to dwell on the disallowed Stamkos goal in the First Period, because ultimately that's not what undid the Lightning in this game. They had a 5-on-3 for a full 2 minutes and only got a slow dribbler chance by Callahan that Holtby held out. Then Paquette loses a faceoff and loses his man. Little thing. Down 1-0. They have plenty of guys back in transition and they completely blow the coverage and Backstrom gets a rebound goal. Little thing. Down 2-0. They have a neutral zone turnover and the defense parts to let Backstrom get a prime chance in the slot. Little thing. Down 3-0. They even have 2 players collide at center with the extra attacker so Fehr can get the EN goal. Little things, and they surrender the 4th goal. It's not like the Caps have dominated these two games, but the Lightning have done everything in their power to make them look like world beaters. Other than the PK there was very little of value to take out of tonight, other than what little morale building you can get out of climbing to withing 1 goal late in a game where you were down 3-0 in the Third Period.
Moving back to basics: Cooper's got to get these guys to get 35 shots on goal a night. The shot totals have been anemic lately, and when shot totals are low you don't get a good diet of greasy goals. They need that, and they need a healthy Palat back. A functional PP would be a nice change of pace, too. Every season has a spot like this where the offense will just struggle to get goals. It seems like, more often than not, the Lightning's struggle seems to come in December. It happened in '03. It's happening now.
Cedric Paquette was -1 with 4 penalty minutes, 3 hits, and he was 33% on draws. The first goal was on him. He lost the draw and then lost Backstrom, which allowed Backstrom to easily collect the blocked shot and roll to the slot to get the backhander. Not good.
Jonathan Drouin was -2 with 2 penalty minutes, 1 shot, and 1 hit in 10:55. He was on the ice for Paquette's faux-pas and the Fehr empty-netter, so the +/- is deceiving. He got more comfortable and looked more dangerous as the game rolled on.
Box score and extended statistics from NHL.com.
NHL Game Night: 12-11-14 Hurricanes at LightningSubmitted by pete on December 11, 2014 - 22:40
Tonight's victory brought to you by Crisco.
Evgeny Nabokov allowed 1 goal on 30 shots for the victory. After some rough sledding early in the season, he's put together quality starts in his last 2 contests and he just evened up his season record at 3-3-1.
First Period
Second Period
Third Period
6:35 TB Kucherov (10), (Stralman, Nabokov)(PP)
12:03 CAR Faulk (5), (Nash, Sekera)
19:29 TB Kucherov (11), (Killorn, Carle)
Nabokov and Nikita Kucherov were the game's first and second stars. Kucherov's two goals were of the extremely greasy variety: one a tap in of a dump in pass that took an odd hop in front of an unattended cage, and the second a pass that dribbled in off the foot of a Carolina defender. Nabokov, for his part, only surrendered one greasy goal off a weak point shot that deflected in off of Victor Hedman. It was one of those kinds of nights.
The Lightning still aren't playing their best hockey. It's not for a lack of effort, but they're just not sharp right now and they actually got outshot by the Canes tonight. Still, the win tonight put them at 13 points for the 10 game segment and they're now sitting on a total of 5 insurance points heading into the 4th 10-game segment of the campaign. There's worst places to be, certainly.
Cedric Paquette had 2 shots and 2 hits and was 60% on draws in 14:18. He had a very active First Period with a couple of quality chances set up by Drouin.
Jonathan Drouin was scoreless in 11:51.
Box score and extended statistics from NHL.com.