The inflammatory proteome, obesity, and medical weight loss and regain in humans.

OBJECTIVE: Weight regain occurs after medical weight loss via mechanisms of post-weight-loss "metabolic adaptation." The relationship of inflammatory proteins with weight loss/regain was studied to determine a role for inflammation in metabolic adaptation. METHODS: Seventy-four proteins central to inflammation and immune regulation (Olink) were analyzed in plasma from up to 490 participants in a trial of medical weight-loss maintenance. Cross-sectional and longitudinal associations of proteins with weight were measured using linear and mixed effects regression models and t testing, with replication in the Framingham Heart Study. RESULTS: Broad changes in the inflammatory proteome were observed among the study cohort (60% women, 35% African American) with initial weight loss of ≈8 kg from a median 94 kg at study entry (33/74 proteins; 7 increased; 26 decreased), many of which tracked with weight regain of median ≈2 kg over the next 30 months. Ten proteins were associated with different rates of weight regain, some specifying pathways of chemotaxis and innate immune responses. Several of the observed protein associations were also linked to prevalent obesity in the Framingham Heart Study. CONCLUSIONS: Broad changes in the inflammatory proteome track with changes in weight and may identify specific pathways that modify patterns of weight regain.

Platelets and platelet-like particles mediate intercellular RNA transfer.

The role of platelets in hemostasis and thrombosis is clearly established; however, the mechanisms by which platelets mediate inflammatory and immune pathways are less well understood. Platelets interact and modulate the function of blood and vascular cells by releasing bioactive molecules. Although the platelet is anucleate, it contains transcripts that may mirror disease. Platelet mRNA is only associated with low-level protein translation; however, platelets have a unique membrane structure allowing for the passage of small molecules, leading to the possibility that its cytoplasmic RNA may be passed to nucleated cells. To examine this question, platelet-like particles with labeled RNA were cocultured with vascular cells. Coculture of platelet-like particles with activated THP-1, monocytic, and endothelial cells led to visual and functional RNA transfer. Posttransfer microarray gene expression analysis of THP-1 cells showed an increase in HBG1/HBG2 and HBA1/HBA2 expression that was directly related to the transfer. Infusion of wild-type platelets into a TLR2-deficient mouse model established in vivo confirmation of select platelet RNA transfer to leukocytes. By specifically transferring green fluorescent protein, we also observed external RNA was functional in the recipient cells. The observation that platelets possess the capacity to transfer cytosolic RNA suggests a new function for platelets in the regulation of vascular homeostasis.

Directing quadruplex-stabilizing drugs to the telomere: synthesis and properties of acridine-oligonucleotide conjugates.

Conjugates containing quadruplex-stabilizing acridines linked to oligonucleotides that are complementary to the G-rich human telomere sequence were synthesized. Acylation of 3,6-diaminoacridine followed by two Michael reactions provided derivatives suitable for conjugation, which were coupled to resin-linked amine-modified oligonucleotides by activating the carboxyl group with pentafluorophenyl 4-nitrobenzenesulfonate. After deprotection with aqueous ammonia at room temperature, conjugates incorporating different acridines, linkers, and oligonucleotide sequences were obtained. These were tested for their ability to stabilize intramolecular DNA quadruplexes that are based on the human telomeric repeat sequence (GGGTTA)(n).

Inosine substitutions demonstrate that intramolecular DNA quadruplexes adopt different conformations in the presence of sodium and potassium.

We have examined the stability of fluorescently-labelled oligonucleotides that are based on the human telomeric repeat [(GGGTTA)(3)GGG], in which one of the guanines in turn is substituted with inosine. We show that the relative stability of the substitutions is different in the presence of sodium and potassium. The data for potassium suggest a parallel arrangement of the strands, while the sodium form is mixed parallel and antiparallel.

Influence of loop size on the stability of intramolecular DNA quadruplexes.

We have determined the stability of intramolecular DNA quadruplexes in which the four G(3)-tracts are connected by non-nucleosidic linkers containing propanediol, octanediol or hexaethylene glycol, replacing the TTA loops in the human telomeric repeat sequence. We find that these sequences all fold to form intramolecular complexes, which are stabilized by lithium < sodium < potassium. Quadruplex stability increases in the order propanediol < hexaethylene glycol < octanediol. The shallower shape of the melting profile with propanediol linkers and its lower dependency on potassium concentration suggests that this complex contains fewer stacks of G-quartets. The sequence with octanediol linkers displays a biphasic melting profile, suggesting that it can adopt more than one stable structure. All these complexes display melting temperatures above 310 K in the presence of 10 mM lithium, without added potassium, in contrast to the telomeric repeat sequence. These complexes also fold much faster than the telomeric repeat and there is little or no hysteresis between their melting and annealing profiles. In contrast, the human telomeric repeat sequence and a complex containing two hexaethylene glycol groups in each loop, are less stable and fold more slowly. The melting and annealing profiles for the latter sequence show significant differences, even when heated at 0.2 degrees C min(-1). CD spectra for the oligonucleotides containing non-nucleosidic linkers show positive maxima at 264 nm, with negative minima approximately 244 nm, which are characteristic of parallel quadruplex structures. These results show that the structure and stability of intramolecular quadruplexes is profoundly influenced by the length and composition of the loops.

The stability of intramolecular DNA quadruplexes with extended loops forming inter- and intra-loop duplexes.

We have examined the formation of intramolecular quadruplex DNA structures in which the loops have been extended so as to generate short DNA duplexes. Fluorescence melting and DNase I cleavage studies show that duplexes can be formed within each loop, but that duplexes between the loops are not stable.

Stability of intramolecular DNA quadruplexes: comparison with DNA duplexes.

We have determined the stability of intramolecular quadruplexes that are formed by a variety of G-rich sequences, using oligonucleotides containing appropriately placed fluorophores and quenchers. The stability of these quadruplexes is compared with that of the DNA duplexes that are formed on addition of complementary C-rich oligonucleotides. We find that the linkers joining the G-tracts are not essential for folding and can be replaced with nonnucleosidic moieties, though their sequence composition profoundly affects quadruplex stability. Although the human telomere repeat sequence d[G(3)(TTAG(3))(3)] folds into a quadruplex structure, this forms a duplex in the presence of the complementary C-rich strand at physiological conditions. The Tetrahymena sequence d[G(4)(T(2)G(4))(3)], the sequence d[G(3)(T(2)G(3))(3)], and sequences related to regions of the c-myc promoter d(G(4)AG(4)T)(2) and d(G(4)AG(3)T)(2) preferentially adopt the quadruplex form in potassium-containing buffers, even in the presence of a 50-fold excess of their complementary C-rich strands, though the duplex predominates in the presence of sodium. The HIV integrase inhibitor d[G(3)(TG(3))(3)] forms an extremely stable quadruplex which is not affected by addition of a 50-fold excess of the complementary C-rich strand in both potassium- and sodium-containing buffers. Replacing the TTA loops of the human telomeric repeat with AAA causes a large decrease in quadruplex stability, though a sequence with AAA in the first loop and TTT in the second and third loops is slightly more stable.

High throughput measurement of duplex, triplex and quadruplex melting curves using molecular beacons and a LightCycler.

We have used oligonucleotides containing molecular beacons to determine melting profiles for intramolecular DNA duplexes, triplexes and quadruplexes (tetraplexes). The synthetic oligonucleotides used in these studies contain a fluorophore (fluorescein) and quencher (methyl red) attached either to deoxyribose or to the 5 position of dU. In the folded DNA structures the fluorophore and quencher are in close proximity and the fluorescence is quenched. When the structures melt, the fluorophore and quencher are separated and there is a large increase in fluorescence. These experiments were performed in a Roche LightCycler; this requires small amounts of material (typically 4 pmol oligonucleotide) and can perform 32 melting profiles in parallel. We have used this technique to compare the stability of triplexes containing different base analogues and to confirm the selectivity of a triplex-binding ligand for triplex, rather than duplex, DNA. We have also compared the melting of inter- and intramolecular quadruplexes.