ASARs are a family of very-long noncoding RNAs that control replication timing on individual human autosomes, and are essential for chromosome stability. The eight known ASAR lncRNAs remain closely associated with their parent chromosomes. Analysis of RNA-protein interaction data (from ENCODE) revealed numerous RBPs with significant interactions with multiple ASAR lncRNAs, with several hnRNPs as abundant interactors. An ~7 kb domain within the ASAR6-141 lncRNA shows a striking density of RBP interaction sites. Genetic deletion and ectopic integration assays indicate that this ~7 kb RNA binding protein domain contains functional sequences for controlling replication timing of entire chromosomes in cis. shRNA-mediated depletion of 10 different RNA binding proteins, including HNRNPA1, HNRNPC, HNRNPL, HNRNPM, HNRNPU, or HNRNPUL1, results in dissociation of ASAR lncRNAs from their chromosome territories, and disrupts the synchronous replication that occurs on all autosome pairs, recapitulating the effect of individual ASAR knockouts on a genome-wide scale. Our results further demonstrate the role that ASARs play during the temporal order of genome-wide replication, and we propose that ASARs function as essential RNA scaffolds for the assembly of hnRNP complexes that help maintain the structural integrity of each mammalian chromosome.

Supplementary Figure 1 from Ionizing Radiation Induces Frequent Translocations with Delayed Replication and Condensation

Supplementary Figure 4 from Ionizing Radiation Induces Frequent Translocations with Delayed Replication and Condensation

Supplementary Figure 3 from Ionizing Radiation Induces Frequent Translocations with Delayed Replication and Condensation

The objective of this study was to explore the effects of three weekly frequency doses of high-intensity functional training (HIFT) on an array of cardiometabolic markers in adults with metabolic syndrome (MetS). Twenty-one men and women, randomized into one (HIFT1), two (HIFT2), or three (HIFT3) days per week of HIFT, completed 3-weeks of familiarization plus a 12-week progressive training program. Pre- and post-intervention, several cardiometabolic, body composition, oxygen consumption, metabolic syndrome severity, and perceptions of fitness measurements were assessed. Additionally, an exercise enjoyment survey was administered post-intervention. A Cohen's d was used to demonstrate within-group change effect size. Although this study was not fully powered, a one-way and two-way ANOVA were used to compare the dose groups to provide provisional insights. No differences were found when frequency dose groups were compared. Many cardiometabolic, body composition, and fitness improvements were seen within each group, with clinically meaningful improvements in the metabolic syndrome severity score (MSSS) (HIFT1: -0.105, d = 0.28; HIFT2: -0.382, d = 1.20; HIFT3: -0.467, d = 1.07), waist circumference (HIFT1: -4.1cm, d = 3.33; HIFT2: -5.4cm, d = 0.89; HIFT3: -0.7cm, d = 0.20), and blood glucose (HIFT1: -9.5mg/dL, d = 0.98; HIFT2: -4.9mg/dL, d = 1.00; HIFT3: -1.7mg/dL, d = 0.23). All three groups similarly reported high exercise enjoyment and likeliness to continue after the intervention. In conclusion, HIFT performed once, twice, or thrice a week elicits improvements in MetS and is considered enjoyable. HIFT, even at a low weekly dose, therefore represents a potential strategy to reduce the global MetS burden.

Supplementary Figure 2 from Ionizing Radiation Induces Frequent Translocations with Delayed Replication and Condensation

Individual variability in the severity of dyslipidemia likely leads to the inconsistency of acute lipid responses to exercise. Our understanding of this inconsistency is limited, as research traditionally does not report individual responses. For optimal individualized exercise prescription to treat dyslipidemia, disclosure of the distinct responses is needed to improve our understanding. Purpose: To demonstrate proof-of-concept by reporting the individual profile, blood lipid and apolipoprotein B (ApoB) responses to a single bout of high intensity functional training (HIFT) in adults with metabolic syndrome (MetS). Methods: Four participants with MetS were studied. After measurement of V02peak, participants underwent three weeks of HIFT familiarization. Following familiarization, participants were tested on three consecutive mornings after a 12 hour fast: day 1 - assessment of resting energy expenditure (EE) and baseline (BL) blood lipids; day 2 - EE of a single HIFT bout, followed by immediate assessment (IPE) of blood lipids; day 3 - assessment of blood lipids 24 hours later (24PE). Results: There was considerable variation in lipid and ApoB responses across all four participants (see Figure 1). For example, percent change in low-density lipoprotein triglyceride from BL to IPE and from IPE to 24PE, ranged from -49% to +53%, and -8% to +32%, respectively. Conclusion: Despite MetS among all participants, blood lipid and ApoB responses varied. Our findings support the concept that exercise prescription to treat dyslipidemia warrants inquiry into individual responses.

High intensity functional training (HIFT) provides a potential option to meet public exercise recommendations for both cardiorespiratory and strength outcomes in a time efficient manner. To better understand the potential for HIFT as an exercise approach, energy expenditure (EE) and relative intensity need quantifying. In thirteen sedentary men and women with metabolic syndrome (MetS), we used both indirect calorimetry and blood lactate levels to calculate EE of a single session of HIFT. The HIFT session included four, 6-minute sets of consecutive functional exercises. Examples of the exercises involved were squats, deadlifts, suspension rows, suspension chest press, and planks. Intensity is described relative to individual ventilatory thresholds. The total group EE was 270.3 ± 77.3 kcal with approximately 5% attributed anaerobic energy production. VO2 ranged between 88.8 ± 12.3% and 99 ± 12% of the second ventilatory threshold (VT2), indicating a vigorous effort. After each work interval, peak blood lactate ranged between 7.9 ± 1.9 and 9.3 ± 2.9 mmol, and rate of perceived exertion between 6.9 ± 1.0 and 8.7 ± 0.8 arbitrary units from 1-10. These were achieved in approximately 46 minutes of exercise per participant. In conclusion, HIFT elicits the energy expenditure and effort requisite to result in the adaptive responses to produce the known suite of benefits of exercise for individuals with MetS.

ASARs are long noncoding RNA genes that control replication timing of entire human chromosomes in cis. The three known ASAR genes are located on human chromosomes 6 and 15, and are essential for chromosome integrity. To identify ASARs on all human chromosomes we utilize a set of distinctive ASAR characteristics that allow for the identification of hundreds of autosomal loci with epigenetically controlled, allele-restricted behavior in expression and replication timing of coding and noncoding genes, and is distinct from genomic imprinting. Disruption of noncoding RNA genes at five of five tested loci result in chromosome-wide delayed replication and chromosomal instability, validating their ASAR activity. In addition to the three known essential cis-acting chromosomal loci, origins, centromeres, and telomeres, we propose that all mammalian chromosomes also contain "Inactivation/Stability Centers" that display allele-restricted epigenetic regulation of protein coding and noncoding ASAR genes that are essential for replication and stability of each chromosome.

DNA replication occurs on mammalian chromosomes in a cell-type distinctive temporal order known as the replication timing program. We previously found that disruption of the noncanonical lncRNA genes ASAR6 and ASAR15 results in delayed replication timing and delayed mitotic chromosome condensation of human chromosomes 6 and 15, respectively. ASAR6 and ASAR15 display random monoallelic expression and display asynchronous replication between alleles that is coordinated with other random monoallelic genes on their respective chromosomes. Disruption of the expressed allele, but not the silent allele, of ASAR6 leads to delayed replication, activation of the previously silent alleles of linked monoallelic genes, and structural instability of human chromosome 6. In this report, we describe a second lncRNA gene (ASAR6-141) on human chromosome 6 that when disrupted results in delayed replication timing in cisASAR6-141 is subject to random monoallelic expression and asynchronous replication and is expressed from the opposite chromosome 6 homolog as ASAR6 ASAR6-141 RNA, like ASAR6 and ASAR15 RNAs, contains a high L1 content and remains associated with the chromosome territory where it is transcribed. Three classes of cis-acting elements control proper chromosome function in mammals: origins of replication, centromeres, and telomeres, which are responsible for replication, segregation, and stability of all chromosomes. Our work supports a fourth type of essential chromosomal element, the "Inactivation/Stability Center," which expresses ASAR lncRNAs responsible for proper replication timing, monoallelic expression, and structural stability of each chromosome.

Introduction: Ischemic preconditioning (IPC), a process of cyclically occluding and reperfusing blood to tissue, has been shown to preserve ATP, prolong vasodilation, and enhance exercise performance. Purpose: The aim of this study was to test the effects of IPC on repeated supramaximal cycling performance and recovery in 12 experienced cyclists. Methods: In a randomized, double-blind, placebo controlled, cross-over design, subjects received a 4 x 5 min bilateral leg IPC treatment then performed a supramaximal cycling trial, followed by a 20 min passive recovery, then repeated the identical trial and recovery again. Time to fatigue (TTF) for each trial was measured as well as blood lactate (La) and pH at minute 0, 5, 10, 15, and 20 during the passive recovery. Results: No significant effect was found for IPC on TTF for trial A (p>.05) and trial B (p>.05), however a great amount of individual variability was observed. No significant effect was found for IPC on the amount of La and pH recovery for trial A (p>.05) and B (p>.05). A significant effect was found earlier on the rate of La recovery in the IPC condition compared to placebo in both trial A (p<.05) and trial B (p<.05). Conclusion: These results suggest there is no effect of IPC on repeated supramaximal cycling performance, nor the amount of blood lactate or pH recovery, however, IPC does have an effect on the rate of blood lactate recovery.

Mammalian cells replicate their chromosomes via a temporal replication program. The ASAR6 and ASAR15 genes were identified as loci that when disrupted result in delayed replication and condensation of entire human chromosomes. ASAR6 and ASAR15 are monoallelically expressed long noncoding RNAs that remain associated with the chromosome from which they are transcribed. The chromosome-wide effects of ASAR6 map to the antisense strand of an L1 retrotransposon within ASAR6 RNA, deletion or inversion of which delayed replication of human chromosome 6. Furthermore, ectopic integration of ASAR6 or ASAR15 transgenes into mouse chromosomes resulted in delayed replication and condensation, an increase in H3K27me3, coating of the mouse chromosome with ASAR RNA, and a loss of mouse Cot-1 RNA expression in cis. Targeting the antisense strand of the L1 within ectopically expressed ASAR6 RNA restored normal replication timing. Our results provide direct evidence that L1 antisense RNA plays a functional role in chromosome-wide replication timing of mammalian chromosomes.

DNA replication initiates at multiple sites along each mammalian chromosome at different times during each S phase, following a temporal replication program. We have used a Cre/loxP-based strategy to identify cis-acting elements that control this replication-timing program on individual human chromosomes. In this report, we show that rearrangements at a complex locus at chromosome 15q24.3 result in delayed replication and structural instability of human chromosome 15. Characterization of this locus identified long, RNA transcripts that are retained in the nucleus and form a "cloud" on one homolog of chromosome 15. We also found that this locus displays asynchronous replication that is coordinated with other random monoallelic genes on chromosome 15. We have named this locus ASynchronous replication and Autosomal RNA on chromosome 15, or ASAR15. Previously, we found that disruption of the ASAR6 lincRNA gene results in delayed replication, delayed mitotic condensation and structural instability of human chromosome 6. Previous studies in the mouse found that deletion of the Xist gene, from the X chromosome in adult somatic cells, results in a delayed replication and instability phenotype that is indistinguishable from the phenotype caused by disruption of either ASAR6 or ASAR15. In addition, delayed replication and chromosome instability were detected following structural rearrangement of many different human or mouse chromosomes. These observations suggest that all mammalian chromosomes contain similar cis-acting loci. Thus, under this scenario, all mammalian chromosomes contain four distinct types of essential cis-acting elements: origins, telomeres, centromeres and "inactivation/stability centers", all functioning to promote proper replication, segregation and structural stability of each chromosome.

PURPOSE: Acute mountain sickness (AMS) is a syndrome commonly experienced in non-acclimatized mountaineers when ascent is too high and too rapid. Ischemic preconditioning (IPC) is a noninvasive experimental technique that has been shown to protect remote organs from ensuing hypoxic damage. In this study we sought to determine if IPC would 1) mitigate the effects of altitude on arterial O2 saturation (SpO2) and 2) attenuate the symptoms of AMS. METHODS: Ten (6 men and 4 women) physically active individuals (Age: 26.7±5.0 yrs, VO2max: 45.0±7.1 mL·kg-1·min-1) who were acclimated to 2350m were randomized to either a treatment (IPC) or control (CON) group. An IPC protocol consisting of 3x5 min bilateral leg occlusion/reperfusion bouts at 200 mmHg was administered to the IPC group. The CON group was administered a protocol identical in time and frequency, but with an inflation pressure of 40 mmHg. To examine the potential late phase protective effects of IPC on SpO2 and symptoms of AMS, 36 hours post-IPC or CON, all participants hiked 9km at a standardized pace to an elevation of 3800m. Symptoms of AMS were evaluated by Lake Louise score (LLS). RESULTS: It was found that SpO2 was significantly higher (p<0.05) in the IPC group when compared to the CON group (IPC 89.6 ± 3.9 % vs. CON 86.9 ± 4.2 %,). A LLQ score of less than 3 is considered mild AMS, whereas a score of 3 or more is considered severe AMS. Incidence of severe AMS was significantly lower (p<0.05) in the IPC (0%) vs. CON (50%) group. Moreover, the IPC group (when compared to CON group) had significantly (p<0.05) lower incidence of dizziness/lightheadedness (IPC=0% vs. CON=50%), fatigue/weakness (IPC=25% vs. CON=66.6%), change in mental status (IPC=0% vs. CON=33.3%) and ataxia (IPC=0% vs. CON=16.6%). CONCLUSION: Our findings suggest that IPC may be a strategy to increase SpO2 and decrease AMS symptoms at high altitude.

Mammalian chromosomes initiate DNA replication at multiple sites along their length during each S phase following a temporal replication program. The majority of genes on homologous chromosomes replicate synchronously. However, mono-allelically expressed genes such as imprinted genes, allelically excluded genes, and genes on female X chromosomes replicate asynchronously. We have identified a cis-acting locus on human chromosome 6 that controls this replication-timing program. This locus encodes a large intergenic non-coding RNA gene named Asynchronous replication and Autosomal RNA on chromosome 6, or ASAR6. Disruption of ASAR6 results in delayed replication, delayed mitotic chromosome condensation, and activation of the previously silent alleles of mono-allelic genes on chromosome 6. The ASAR6 gene resides within an ∼1.2 megabase domain of asynchronously replicating DNA that is coordinated with other random asynchronously replicating loci along chromosome 6. In contrast to other nearby mono-allelic genes, ASAR6 RNA is expressed from the later-replicating allele. ASAR6 RNA is synthesized by RNA Polymerase II, is not polyadenlyated, is restricted to the nucleus, and is subject to random mono-allelic expression. Disruption of ASAR6 leads to the formation of bridged chromosomes, micronuclei, and structural instability of chromosome 6. Finally, ectopic integration of cloned genomic DNA containing ASAR6 causes delayed replication of entire mouse chromosomes.

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation(1). Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of >3 hours that affects the entire chromosome(2,3). Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome(4). Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes(5).Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from(6). In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Mammalian DNA replication initiates at multiple sites along chromosomes at different times, following a temporal replication program. Homologous alleles typically replicate synchronously; however, mono-allelically expressed genes such as imprinted genes, allelically excluded genes and genes on the female X chromosome replicate asynchronously. We have used a chromosome engineering strategy to identify a human autosomal locus that controls this replication timing program in cis. We show that Cre/loxP-mediated rearrangements at a discrete locus at 6q16.1 result in delayed replication of the entire chromosome. This locus displays asynchronous replication timing that is coordinated with other mono-allelically expressed genes on chromosome 6. Characterization of this locus revealed mono-allelic expression of a large intergenic non-coding RNA, which we have named asynchronous replication and autosomal RNA on chromosome 6, ASAR6. Finally, disruption of this locus results in the activation of the previously silent alleles of linked mono-allelically expressed genes. We previously found that chromosome rearrangements involving eight different autosomes display delayed replication timing, and that cells containing chromosomes with delayed replication timing have a 30-80-fold increase in the rate at which new gross chromosomal rearrangements occurred. Taken together, these observations indicate that human autosomes contain discrete cis-acting loci that control chromosome-wide replication timing, mono-allelic expression and the stability of entire chromosomes.

Murine hepatocytes become polyploid and then undergo ploidy reversal and become aneuploid in a dynamic process called the ploidy conveyor. Although polyploidization occurs in some types of human cells, the degree of aneuploidy in human hepatocytes is not known. We isolated hepatocytes derived from healthy human liver samples and determined chromosome number and identity using traditional karyotyping and fluorescence in situ hybridization. Similar to murine hepatocytes, human hepatocytes are highly aneuploid. Moreover, imaging studies revealed multipolar spindles and chromosome segregation defects in dividing human hepatocytes. Aneuploidy therefore does not necessarily predispose liver cells to transformation but might promote genetic diversity among hepatocytes.

Certain chromosome rearrangements display a significant delay in chromosome replication timing (DRT) that is associated with a subsequent delay in mitotic chromosome condensation (DMC). DRT/DMC chromosomes are common in tumor cells in vitro and in vivo and occur frequently in cells exposed to ionizing radiation. A hallmark for these chromosomes is the delayed phosphorylation of serine 10 of histone H3 during mitosis. The chromosome passenger complex, consisting of multiple proteins including Aurora B kinase and INCENP is thought to be responsible for H3 phosphorylation, chromosome condensation and the subsequent segregation of chromosomes. In this report, we show that chromosomes with DRT/DMC contain phosphorylated Chk1, consistent with activation of the S–M phase checkpoint. Furthermore, we show that INCENP is recruited to the DRT/DMC chromosomes during all phases of mitosis. In contrast, Aurora B kinase is absent on DRT/DMC chromosomes when these chromosomes lack serine 10 phosphorylation of H3. We also show that mitotic arrest deficient 2 (Mad2), a member of the spindle assembly checkpoint, is present on DRT/DMC chromosomes at a time when the normally condensed chromosomes show no Mad2 staining, indicating that DRT/DMC activates the spindle assembly checkpoint. Finally, cells with DRT/DMC chromosomes have centrosome amplification, abnormal spindle assembly, endoreduplication and significant chromosome instability.