Cot-based sequence discovery represents a powerful means by which both low-copy and repetitive sequences can be selectively and efficiently fractionated, cloned, and characterized. Based upon the results of a Cot analysis, hydroxyapatite chromatography was used to fractionate sorghum (Sorghum bicolor) genomic DNA into highly repetitive (HR), moderately repetitive (MR), and single/low-copy (SL) sequence components that were consequently cloned to produce HRCot, MRCot, and SLCot genomic libraries. Filter hybridization (blotting) and sequence analysis both show that the HRCot library is enriched in sequences traditionally found in high-copy number (e.g., retroelements, rDNA, centromeric repeats), the SLCot library is enriched in low-copy sequences (e.g., genes and “nonrepetitive ESTs”), and the MRCot library contains sequences of moderate redundancy. The Cot analysis suggests that the sorghum genome is approximately 700 Mb (in agreement with previous estimates) and that HR, MR, and SL components comprise 15%, 41%, and 24% of sorghum DNA, respectively. Unlike previously described techniques to sequence the low-copy components of genomes, sequencing of Cot components is independent of expression and methylation patterns that vary widely among DNA elements, developmental stages, and taxa. High-throughput sequencing of Cot clones may be a means of “capturing” the sequence complexity of eukaryotic genomes at unprecedented efficiency.[Online supplementary material is available at www.genome.org. The sequence data described in this paper have been submitted to the GenBank under accession nos.AZ921847-AZ923007. Reagents, samples, and unpublished information freely provided by H. Ma and J. Messing.]

Research on the cell fate determination of embryonic stem cells is of enormous interest given the therapeutic potential in regenerative cell therapy. Human embryonic stem cells (hESCs) have the ability to renew themselves and differentiate into all three germ layers. The main focus of this study was to examine factors affecting derivation and further proliferation of multipotent neuroepithelial (NEP) cells from hESCs. hESCs cultured in serum-deprived defined medium developed distinct tube structures and could be isolated either by dissociation or adherently. Dissociated cells survived to form colonies of cells characterized as NEP when conditioned medium from human hepatocellular carcinoma HepG2 cell line (MEDII) was added. However, cells isolated adherently developed an enriched population of NEP cells independent of MEDII medium. Further characterization suggested that they were NEP cells because they had a similar phenotype profile to in vivo NEP cells and expression SOX1, SOX2, and SOX3 genes. They were positive for Nestin, a neural intermediate filament protein, and Musashi-1, a neural RNA-binding protein, but few cells expressed further differentiation markers, such as PSNCAM, A2B5, MAPII, GFAP, or O4, or other lineage markers, such as muscle actin, α fetoprotein, or the pluripotent marker Oct4. Further differentiation of these putative NEP cells gave rise to a mixed population of progenitors that included A2B5-positive and PSNCAM-positive cells and postmitotic neurons and astrocytes. To proliferate and culture these derived NEP cells, ideal conditions were obtained using neurobasal medium supplemented with B27 and basic fibroblast growth factor in 5% oxygen. NEP cells were continuously propagated for longer than 6 months without losing their multipotent cell characteristics and maintained a stable chromosome number.

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Overexpression of the c-Myc oncoprotein is observed in a large number of hematopoietic malignancies, and transgenic animal models have revealed a potent role for c-Myc in the generation of leukemias and lymphomas. However, the reason for high c-Myc protein levels in most cases is unknown. We examined whether aberrant protein stabilization could be a mechanism of c-Myc overexpression in leukemia cell lines and in primary bone marrow samples from pediatric acute lymphoblastic leukemia (ALL) patients. We found that c-Myc protein half-life was prolonged in the majority of leukemia cell lines and bone marrow samples tested. There were no mutations in the c-myc gene in any of the leukemia cell lines that could account for increased c-Myc stability. However, abnormal phosphorylation at two conserved sites, Threonine 58 and Serine 62, was observed in leukemia cell lines with stabilized c-Myc. Moreover, stabilized c-Myc from the ALL cell lines showed decreased affinity for glycogen synthase kinase3β, the kinase that phosphorylates c-Myc at Threonine 58 and facilitates its degradation. These findings reveal that deregulation of the c-Myc degradation pathway controlled by Serine 62 and Threonine 58 phosphorylation is a novel mechanism for increased expression of a potent oncoprotein known to be involved in hematopoietic malignancies.

Oxidative stress and muscle damage occur during exhaustive bouts of exercise, and many runners report pain and soreness as major influences on changes or breaks in training regimens, creating a barrier to training persistence. Methylsulfonylmethane (MSM) is a sulfur-based nutritional supplement that is purported to have pain and inflammation-reducing effects. To investigate the effects of MSM in attenuating damage associated with physical exertion, this randomized, double-blind, placebo-controlled study evaluated the effects of MSM supplementation on exercise-induced pain, oxidative stress and muscle damage. Twenty-two healthy females (n = 17) and males (n = 5) (age 33.7 ± 6.9 yrs.) were recruited from the 2014 Portland Half-Marathon registrant pool. Participants were randomized to take either MSM (OptiMSM®) (n = 11), or a placebo (n = 11) at 3 g/day for 21 days prior to the race and for two days after (23 total). Participants provided blood samples for measurement of markers of oxidative stress, and completed VAS surveys for pain approximately one month prior to the race (T0), and at 15 min (T1), 90 min (T2), 1 Day (T3), and 2 days (T4) after race finish. The primary outcome measure 8-hydroxy-2-deoxyguanine (8-OHdG) measured oxidative stress. Secondary outcomes included malondialdehyde (MDA) for oxidative stress, creatine kinase (CK) and lactate dehydrogenase (LDH) as measures of muscle damage, and muscle (MP) and joint pain (JP) recorded using a 100 mm Visual Analogue Scale (VAS). Data were analyzed using repeated and multivariate ANOVAs, and simple contrasts compared post-race time points to baseline, presented as mean (SD) or mean change (95% CI) where appropriate. Running a half-marathon induced significant increases in all outcome measures (p < 0.001). From baseline, 8-OHdG increased significantly at T1 by 1.53 ng/mL (0.86–2.20 ng/mL CI, p < 0.001) and T2 by 1.19 ng/mL (0.37–2.01 ng/mL CI, p < 0.01), and fell below baseline levels at T3 by −0.46 ng/mL (−1.18–0.26 CI, p > 0.05) and T4 by −0.57 ng/mL (−1.27–0.13 CI, p > 0.05). MDA increased significantly at T1 by 7.3 μM (3.9–10.7 CI, p < 0.001). Muscle damage markers CK and LDH saw significant increases from baseline at all time-points (p < 0.01). Muscle and joint pain increased significantly from baseline at T1, T2, and T3 (p < 0.01) and returned to baseline levels at T4. Time-by-treatment results did not reach statistical significance for any outcome measure, however, the MSM group saw clinically significant (Δ > 10 mm) reductions in both muscle and joint pain. Participation in a half-marathon was associated with increased markers of oxidative stress, muscle damage, and pain. MSM supplementation was not associated with a decrease from pre-training levels of oxidative stress or muscle damage associated with an acute bout of exercise. MSM supplementation attenuated post-exercise muscle and joint pain at clinically, but not statistically significant levels.

Combined targeting of SET and tyrosine kinases provides an effective therapeutic approach in human T-cell acute lymphoblastic leukemia