Mammalian Comparative Sequence Analysis of the Agrp Locus

Agouti-related protein encodes a neuropeptide that stimulates food intake. Agrp expression in the brain is restricted to neurons in the arcuate nucleus of the hypothalamus and is elevated by states of negative energy balance. The molecular mechanisms underlying Agrp regulation, however, remain poorly defined. Using a combination of transgenic and comparative sequence analysis, we have previously identified a 760 bp conserved region upstream of Agrp which contains STAT binding elements that participate in Agrp transcriptional regulation. In this study, we attempt to improve the specificity for detecting conserved elements in this region by comparing genomic sequences from 10 mammalian species. Our analysis reveals a symmetrical organization of conserved sequences upstream of Agrp, which cluster into two inverted repeat elements. Conserved sequences within these elements suggest a role for homeodomain proteins in the regulation of Agrp and provide additional targets for functional evaluation

Treatment Response Assessment in IDH-Mutant Glioma Patients by Noninvasive 3D Functional Spectroscopic Mapping of 2-Hydroxyglutarate

Purpose: Measurements of objective response rates are critical to evaluate new glioma therapies. The hallmark metabolic alteration in gliomas with mutant isocitrate dehydrogenase (IDH) is the overproduction of oncometabolite 2-hydroxyglutarate (2HG), which plays a key role in malignant transformation. 2HG represents an ideal biomarker to probe treatment response in IDH-mutant glioma patients, and we hypothesized a decrease in 2HG levels would be measureable by in vivo magnetic resonance spectroscopy (MRS) as a result of antitumor therapy. Experimental Design: We report a prospective longitudinal imaging study performed in 25 IDH-mutant glioma patients receiving adjuvant radiation and chemotherapy. A newly developed 3D MRS imaging was used to noninvasively image 2HG. Paired Student t test was used to compare pre- and posttreatment tumor 2HG values. Test-retest measurements were performed to determine the threshold for 2HG functional spectroscopic maps (fSM). Univariate and multivariate regression were performed to correlate 2HG changes with Karnofsky performance score (KPS). Results: We found that mean 2HG (2HG/Cre) levels decreased significantly (median=48.1%; 95% confidence interval=27.3%-56.5%; P=0.007) in the posttreatment scan. The volume of decreased 2HG correlates (R2=0.88, P=0.002) with clinical status evaluated by KPS. Conclusions: We demonstrate that dynamic measurements of 2HG are feasible by 3D fSM, and the decrease of 2HG levels can monitor treatment response in patients with IDH-mutant gliomas. Our results indicate that quantitative in vivo 2HG imaging maybe used for precision medicine and early response assessment in clinical trials of therapies targeting IDH-mutant gliomas

Cytotoxic T lymphocytes that recognize decameric peptide sequences of retinoblastoma binding protein 1 (RBP-1) associated with human breast cancer

Retinoblastoma binding protein 1 (RBP-1) is a 143-kDa nuclear phosphoprotein that promotes cell growth by inhibiting the product of retinoblastoma tumour suppressor gene (pRB). We recently found that RBP-1 contains KASIFLK, a heptameric peptide (250–256) recognized by human antibodies and overexpressed by breast cancer cells. In the present study, we demonstrate that human T-cells stimulated with RBP-1 decameric peptides containing KASIFLK can kill human breast cancer cells. These decamers, GLQKASIFLK (247–256) and KASIFLKTRV (250–259), have anchor motifs for both HLA-A2 and HLA-A3. Peripheral blood lymphocytes from 41 normal donors were stimulated by these peptides in culture media containing 15 IU ml−1 interleukin-2, 25 IU ml−1 interleukin-7 and 500 IU ml−1 granulocyte–macrophage colony-stimulating factor. Cytotoxic activity of the T-cells was assessed against autologous B lymphoblastoid cells pulsed with each peptide. Stimulation by GLQKASIFLK generated specific cytotoxic T lymphocyte (CTL) lines from HLA-A2, A3 donors, HLA-A2 donors and HLA-A3 donors. Stimulation with KASIFLKTRV generated specific CTL lines from HLA-A2 donors. No HLA-A2−, A3− CTL line showed specific cytotoxicity against these target cells. These CTL lines were also cytotoxic against HLA-A2 and HLA-A3 breast cancer cells but not against normal fibroblastoid cell lines, normal epidermal cell lines, or a melanoma cell line. RBP-1 peptide antigens may be of clinical significance as a potential peptide vaccine against human breast cancer. © 1999 Cancer Research Campaig

Pre-training Skeletal Muscle Fiber Size and Predominant Fiber Type Best Predict Hypertrophic Responses to 6 Weeks of Resistance Training in Previously Trained Young Men

Limited evidence exists regarding differentially expressed biomarkers between previously-trained low versus high hypertrophic responders in response to resistance training. Herein, 30 college-aged males (training age 5 ± 3 years; mean ± SD) partook in 6 weeks of high-volume resistance training. Body composition, right leg vastus lateralis (VL) biopsies, and blood were obtained prior to training (PRE) and at the 3-week (W3) and 6-week time points (W6). The 10 lowest (LOW) and 10 highest (HIGH) hypertrophic responders were clustered based upon a composite hypertrophy score of PRE-to-W6 changes in right leg VL mean muscle fiber cross-sectional area (fCSA), VL thickness assessed via ultrasound, upper right leg lean soft tissue mass assessed via dual x-ray absorptiometry (DXA), and mid-thigh circumference. Two-way ANOVAs were used to compare biomarker differences between the LOW and HIGH clusters over time, and stepwise linear regression was performed to elucidate biomarkers that explained significant variation in the composite hypertrophy score from PRE to W3, W3 to W6, and PRE to W6 in all 30 participants. PRE-to-W6 HIGH and LOW responders exhibited a composite hypertrophy change of +10.7 ± 3.2 and -2.1 ± 1.6%, respectively (p < 0.001). Compared to HIGH responders, LOW responders exhibited greater PRE type II fCSA (+18%, p = 0.022). Time effects (p < 0.05) existed for total RNA/mg muscle (W6 > W3 > PRE), phospho (p)-4EBP1 (PRE > W3&W6), pan-mTOR (PRE > W3 < W6), p-mTOR (PRE > W3 < W6), pan-AMPKα (PRE > W3 < W6), pan-p70s6k (PRE > W3), muscle ubiquitin-labeled proteins (PRE > W6), mechano growth factor mRNA (W6 > W3&PRE), 45S rRNA (PRE > W6), and muscle citrate synthase activity (PRE > W3&W6). No interactions existed for the aforementioned biomarkers and/or other assayed targets (muscle 20S proteasome activity, serum total testosterone, muscle androgen receptor protein levels, muscle glycogen, or serum creatine kinase). Regression analysis indicated PRE type II fiber percentage (R2 = 0.152, β = 0.390, p = 0.033) and PRE type II fCSA (R2 = 0.207, β = -0.455, p = 0.019) best predicted the PRE-to-W6 change in the composite hypertrophy score. While our sample size is limited, these data suggest: (a) HIGH responders may exhibit more growth potential given that they possessed lower PRE type II fCSA values and (b) possessing a greater type II fiber percentage as a trained individual may be advantageous for hypertrophy in response to resistance training

Prisoner's Dilemma in Cancer Metabolism

As tumors outgrow their blood supply and become oxygen deprived, they switch to less energetically efficient but oxygen-independent anaerobic glucose metabolism. However, cancer cells maintain glycolytic phenotype even in the areas of ample oxygen supply (Warburg effect). It has been hypothesized that the competitive advantage that glycolytic cells get over aerobic cells is achieved through secretion of lactic acid, which is a by-product of glycolysis. It creates acidic microenvironment around the tumor that can be toxic to normal somatic cells. This interaction can be seen as a prisoner's dilemma: from the point of view of metabolic payoffs, it is better for cells to cooperate and become better competitors but neither cell has an incentive to unilaterally change its metabolic strategy. In this paper a novel mathematical technique, which allows reducing an otherwise infinitely dimensional system to low dimensionality, is used to demonstrate that changing the environment can take the cells out of this equilibrium and that it is cooperation that can in fact lead to the cell population committing evolutionary suicide

Bovine Milk Extracellular Vesicles (EVs) Modification Elicits Skeletal Muscle Growth in Rats

The current study investigated how bovine milk extracellular vesicles (EVs) affected rotarod performance and biomarkers of skeletal muscle physiology in young, growing rats. Twenty-eight-day Fisher 344 rats were provided an AIN-93G-based diet for 4 weeks that either remained unadulterated [EVs and RNA-sufficient (ERS; n = 12)] or was sonicated [EVs and RNA-depleted (ERD; n = 12)]. Prior to (PRE) and on the last day of the intervention (POST), animals were tested for maximal rotarod performance. Following the feeding period, the gastrocnemius muscle was analyzed at the histological, biochemical, and molecular levels and was also used to measure mitochondrial function and reactive oxygen species (ROS) emission. A main effect of time was observed for rotarod time (PRE \u3e POST, p = 0.001). Terminal gastrocnemius mass was unaffected by diet, although gastrocnemius muscle fiber cross sectional area was 11% greater (p = 0.018) and total RNA (a surrogate of ribosome density) was 24% greater (p = 0.001) in ERD. Transcriptomic analysis of the gastrocnemius indicated that 22 mRNAs were significantly greater in ERS versus ERD (p \u3c 0.01), whereas 55 mRNAs were greater in ERD versus ERS (p \u3c 0.01). There were no differences in gastrocnemius citrate synthase activity or mitochondrial coupling (respiratory control ratio), although mitochondrial ROS production was lower in ERD gastrocnemius (p = 0.016), which may be explained by an increase in glutathione peroxidase protein levels (p = 0.020) in ERD gastrocnemius. Dietary EVs profiling confirmed that sonication in the ERD diet reduced EVs content by ∼60%. Our findings demonstrate that bovine milk EVs depletion through sonication elicits anabolic and transcriptomic effects in the gastrocnemius muscle of rapidly maturing rats. While this did not translate into a functional outcome between diets (i.e., rotarod performance), longer feeding periods may be needed to observe such functional effects

Synthetic lethality: a framework for the development of wiser cancer therapeutics

The challenge in medical oncology has always been to identify compounds that will kill, or at least tame, cancer cells while leaving normal cells unscathed. Most chemotherapeutic agents in use today were selected primarily for their ability to kill rapidly dividing cancer cells grown in cell culture and in mice, with their selectivity determined empirically during subsequent animal and human testing. Unfortunately, most of the drugs developed in this way have relatively low therapeutic indices (low toxic dose relative to the therapeutic dose). Recent advances in genomics are leading to a more complete picture of the range of mutations, both driver and passenger, present in human cancers. Synthetic lethality provides a conceptual framework for using this information to arrive at drugs that will preferentially kill cancer cells relative to normal cells. It also provides a possible way to tackle 'undruggable' targets. Two genes are synthetically lethal if mutation of either gene alone is compatible with viability but simultaneous mutation of both genes leads to death. If one is a cancer-relevant gene, the task is to discover its synthetic lethal interactors, because targeting these would theoretically kill cancer cells mutant in the cancer-relevant gene while sparing cells with a normal copy of that gene. All cancer drugs in use today, including conventional cytotoxic agents and newer 'targeted' agents, target molecules that are present in both normal cells and cancer cells. Their therapeutic indices almost certainly relate to synthetic lethal interactions, even if those interactions are often poorly understood. Recent technical advances enable unbiased screens for synthetic lethal interactors to be undertaken in human cancer cells. These approaches will hopefully facilitate the discovery of safer, more efficacious anticancer drugs that exploit vulnerabilities that are unique to cancer cells by virtue of the mutations they have accrued during tumor progression

An intron variant of the GLI family zinc finger 3 (GLI3) gene differentiates resistance training-induced muscle fiber hypertrophy in younger men

We examined the association between genotype and resistance training-induced changes (12 wk) in dual x-ray energy absorptiometry (DXA)-derived lean soft tissue mass (LSTM) as well as muscle fiber cross-sectional area (fCSA; vastus lateralis; n = 109; age = 22 ± 2 y, BMI = 24.7 ± 3.1 kg/m2). Over 315 000 genetic polymorphisms were interrogated from muscle using DNA microarrays. First, a targeted investigation was performed where single nucleotide polymorphisms (SNP) identified from a systematic literature review were related to changes in LSTM and fCSA. Next, genome-wide association (GWA) studies were performed to reveal associations between novel SNP targets with pre- to post-training change scores in mean fCSA and LSTM. Our targeted investigation revealed no genotype-by-time interactions for 12 common polymorphisms regarding the change in mean fCSA or change in LSTM. Our first GWA study indicated no SNP were associated with the change in LSTM. However, the second GWA study indicated two SNP exceeded the significance level with the change in mean fCSA (P = 6.9 × 10–7 for rs4675569, 1.7 × 10–6 for rs10263647). While the former target is not annotated (chr2:205936846 (GRCh38.p12)), the latter target (chr7:41971865 (GRCh38.p12)) is an intron variant of the GLI Family Zinc Finger 3 (GLI3) gene. Follow-up analyses indicated fCSA increases were greater in the T/C and C/C GLI3 genotypes than the T/T GLI3 genotype (P \u3c.05). Data from the Auburn cohort also revealed participants with the T/C and C/C genotypes exhibited increases in satellite cell number with training (P \u3c.05), whereas T/T participants did not. Additionally, those with the T/C and C/C genotypes achieved myonuclear addition in response to training (P \u3c.05), whereas the T/T participants did not. In summary, this is the first GWA study to examine how polymorphisms associate with the change in hypertrophy measures following resistance training. Future studies are needed to determine if the GLI3 variant differentiates hypertrophic responses to resistance training given the potential link between this gene and satellite cell physiology

ER-Alpha-cDNA As Part of a Bicistronic Transcript Gives Rise to High Frequency, Long Term, Receptor Expressing Cell Clones

Within the large group of Estrogen Receptor alpha (ERα)-negative breast cancer patients, there is a subgroup carrying the phenotype ERα−, PR−, and Her2−, named accordingly “Triple-Negative” (TN). Using cell lines derived from this TN group, we wished to establish cell clones, in which ERα is ectopically expressed, forming part of a synthetic lethality screening system. Initially, we generated cell transfectants expressing a mono-cistronic ERα transcription unit, adjacent to a separate dominant selectable marker transcription unit. However, the yield of ERα expressing colonies was rather low (5–12.5%), and only about half of these displayed stable ectopic ERα expression over time. Generation and maintenance of such cell clones under minimal exposure to the ERα ligand, did not improve yield or expression stability. Indeed, other groups have also reported grave difficulties in obtaining ectopic expression of ERα in ERα-deficient breast carcinoma cells. We therefore switched to transfecting these cell lines with pERα-IRES, a plasmid vector encoding a bicistronic translation mRNA template: ERα Open Reading Frame (ORF) being upstream followed by a dominant-positive selectable marker (hygroR) ORF, directed for translation from an Internal Ribosome Entry Site (IRES). Through usage of this bicistronic vector linkage system, it was possible to generate a very high yield of ERα expressing cell clones (50–100%). The stability over time of these clones was also somewhat improved, though variations between individual cell clones were evident. Our successful experience with ERα in this system may serve as a paradigm for other genes where ectopic expression meets similar hardships