BackgroundMetabolic syndrome (MS) is a construct used to separate “healthy” from “unhealthy” obese patients, and is a major risk factor for type 2 diabetes (T2D) and cardiovascular disease. There is controversy over whether obese “metabolically well” persons have a higher morbidity and mortality than lean counterparts, suggesting that MS criteria do not completely describe physiologic risk factors or consequences of obesity. We hypothesized that metabolomic analysis of plasma would distinguish obese individuals with and without MS and T2D along a spectrum of obesity-associated metabolic derangements, supporting metabolomic analysis as a tool for a more detailed assessment of metabolic wellness than currently used MS criteria.MethodsFasting plasma samples from 90 adults were assigned to groups based on BMI and ATP III criteria for MS: (1) lean metabolically well (LMW; n = 24); (2) obese metabolically well (OBMW; n = 26); (3) obese metabolically unwell (OBMUW; n = 20); and (4) obese metabolically unwell with T2D (OBDM; n = 20). Forty-one amino acids/dipeptides, 33 acylcarnitines and 21 ratios were measured. Obesity and T2D effects were analyzed by Wilcoxon rank-sum tests comparing obese nondiabetics vs LMW, and OBDM vs nondiabetics, respectively. Metabolic unwellness was analyzed by Jonckheere-Terpstra trend tests, assuming worsening health from LMW → OBMW → OBMUW. To adjust for multiple comparisons, statistical significance was set at p < 0.005. K-means cluster analysis of aggregated amino acid and acylcarnitine data was also performed.ResultsAnalytes and ratios significantly increasing in obesity, T2D, and with worsening health include: branched-chain amino acids (BCAAs), cystine, alpha-aminoadipic acid, phenylalanine, leucine + lysine, and short-chain acylcarnitines/total carnitines. Tyrosine, alanine and propionylcarnitine increase with obesity and metabolic unwellness. Asparagine and the tryptophan/large neutral amino acid ratio decrease with T2D and metabolic unwellness. Malonylcarnitine decreases in obesity and 3-OHbutyrylcarnitine increases in T2D; neither correlates with unwellness. Cluster analysis did not separate subjects into discreet groups based on metabolic wellness.DiscussionLevels of 15 species and metabolite ratios trend significantly with worsening metabolic health; some are newly recognized. BCAAs, aromatic amino acids, lysine, and its metabolite, alpha-aminoadipate, increase with worsening health. The lysine pathway is distinct from BCAA metabolism, indicating that biochemical derangements associated with MS involve pathways besides those affected by BCAAs. Even those considered “obese, metabolically well” had metabolite levels which significantly trended towards those found in obese diabetics. Overall, this analysis yields a more granular view of metabolic wellness than the sole use of cardiometabolic MS parameters. This, in turn, suggests the possible utility of plasma metabolomic analysis for research and public health applications.
Expression of the genome requires RNA polymerase II (RNAPII) to transcribe across many natural and unnatural barriers, and this transcription across barriers is facilitated by protein complexes called elongation factors (EFs). Genetic studies in Saccharomyces cerevisiae yeast suggest that multiple EFs collaborate to assist RNAPII in completing the transcription of genes, but the molecular mechanisms of how they cooperate to promote elongation are not well understood. The Ccr4-Not complex participates in multiple steps of mRNA metabolism and has recently been shown to be an EF. Here we describe how Ccr4-Not and TFIIS cooperate to stimulate elongation. We find that Ccr4-Not and TFIIS mutations show synthetically enhanced phenotypes, and biochemical analyses indicate that Ccr4-Not and TFIIS work synergistically to reactivate arrested RNAPII. Ccr4-Not increases the recruitment of TFIIS into elongation complexes and enhances the cleavage of the displaced transcript in backtracked RNAPII. This is mediated by an interaction between Ccr4-Not and the N terminus of TFIIS. In addition to revealing insights into how these two elongation factors cooperate to promote RNAPII elongation, our study extends the growing body of evidence suggesting that the N terminus of TFIIS acts as a docking/interacting site that allows it to synergize with other EFs to promote RNAPII transcription.T ranscription of genes by RNA polymerase II (RNAPII) is a well-orchestrated process that involves steps of initiation, elongation, and termination. Following promoter clearance, RNAPII enters the phase of productive elongation that is achieved by a Brownian ratchet mechanism, in which the RNAPII oscillates between a pretranslocated and a posttranslocated state. After nucleotide addition to the 3= end of the RNA, the incoming nucleoside triphosphate (NTP) locks RNAPII in a posttranslocated form, readying it for the next cycle (1-4). However, productive elongation is not a product of efficient addition of nucleotides by RNAPII alone. During transcription elongation, RNAPII encounters several blocks, including sequence-specific pause sites, nucleotide limitations, DNA lesions, negative elongation factors, and DNA-bound proteins, which cause RNAPII to pause, arrest, or terminate transcription (5, 6). A myriad of elongation factors helps rescue paused/arrested polymerases and stimulate transcription (7,8). Each elongation factor acts via a different mechanism, and often, one works in combination with others.One of the best-characterized elongation factors known to rescue backtracked RNAPII is TFIIS. TFIIS promotes transcription elongation by stimulating the nucleolytic activity of RNAPII and realigning the 3= end of the transcript in the active site of arrested RNAPII (for reviews, see references 9 and 10). New evidence suggests that the cleavage-promoting activity of TFIIS is not the only way in which it stimulates elongation (11-13). Biochemical and biophysical studies have only recently begun to uncover the mechanisms by which TFIIS functions with TFII...
In stratified epithelia such as the epidermis, homeostasis is maintained by the proliferation of cells in the lower epithelial layers and the concomitant loss of differentiated cells from the epithelial surface. These differentiating keratinocytes progressively stratify and form a self‐regenerating multi‐layered barrier that protects the underlying dermis. In such tissue, the continual loss and replacement of differentiated cells also limits the accumulation of oncogenic mutations within the tissue. Inactivating mutations in key driver genes, such as TP53 and NOTCH1, reduce the proportion of differentiating cells allowing for the long‐term persistence of expanding mutant clones in the tissue. Here we show that through the expression of E6, HPV‐16 prevents the early fate commitment of human keratinocytes towards differentiation and confers a strong growth advantage to human keratinocytes. When E6 is expressed either alone or with E7, it promotes keratinocyte proliferation at high cell densities, through the combined inactivation of p53 and Notch1. In organotypic raft culture, the activity of E6 is restricted to the basal layer of the epithelium and is enhanced during the progression from productive to abortive or transforming HPV‐16 infection. Consistent with this, the expression of p53 and cleaved Notch1 becomes progressively more disrupted, and is associated with increased basal cell density and reduced commitment to differentiation. The expression of cleaved Notch1 is similarly disrupted also in HPV‐16‐positive cervical lesions, depending on neoplastic grade. When taken together, these data depict an important role of high‐risk E6 in promoting the persistence of infected keratinocytes in the basal and parabasal layers through the inactivation of gene products that are commonly mutated in non‐HPV‐associated neoplastic squamous epithelia. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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