Glycosides

Backbone parameters comprise the single bond torsions along the phosphodiester chain and the conformation of the sugar ring. In a conventional DNA strand, the backbone segment associated with each nucleotide (in the 5′→3′ direction) is described by the torsions α (03′-P-O5′-C5′), β (P-O5′-C5′-C4′), γ (O5′-C5′-C4′-C3′), δ (C5′-C4′-C3′-O3′), ϵ (C4′-C3′-O3′-P) and ζ (C3′-O3′-P-O5′), to which we must add the glycosidic angle χ (O4′-C1′-N1-C2 for pyrimidines and O4′-C1′-N9-C4 for purines) joining the sugar to the base and the ribose OH torsion (C1′-C2′-O2′-H2′) in the case of RNA.
We remark that calculating averages and standard deviations of angular variables is not trivial, unless they cover restricted angular ranges. There is also no simple definition of maximal and minimal values. This problem occurs in many branches of science with broadly distributed angular variables, for example, in analysing wind directions (30 ). While angular helical variables generally lie within limited ranges, backbone dihedrals can easily span the full range of 360°. In this case, maximal and minimal values in the Curves+ analysis are replaced with the parameter ‘range’ and angular averages and standard deviations are calculated using a vectorial approach. Range is defined as the number of 1° bins visited by a given variable in the interval 0–360°. This gives a good idea of the angular spread of variables. Note that when analysing molecular dynamics trajectories, this value may increase with sampling, giving an indication that more sampling probably needs to be done. However, the details of the angular distribution can be checked using the histogram output option of the supplementary program Canal (see below). For averages, angles are added as vectors in 2D space (with an angle θ having components x = Cos θ and y = Sin θ). The result is converted to a unit vector, whose X and Y components yield the average. Other approaches require assuming that the angles obey a presupposed type of distribution. We have checked our values against one such model (31 ), and found negligible differences for standard deviations up to roughly 20°. Larger values differ more significantly (5–10°), but in these cases it is the qualitative result that the variables in question fluctuate very strongly that is the most important.
The sugar ring is usefully described using pseudorotation parameters. Although strictly speaking there are four pseudorotation parameters for a five-membered ring (32 (link)), only two of these, the so-called phase (Pha) and amplitude (Amp), are generally useful. While the amplitude describes the degree of ring puckering, the phase describes which atoms are most displaced from the mean ring plane. We calculate these parameters using the formulae given below (33 ), which have the advantage of treating the ring dihedrals ν₁ (C1′-C2′-C3′-C4′) to ν₅ (O4′-C1′-C2′-C3′) in an equivalent manner. In this approach:

where and b =−0.4 note, if then .
Conventionally, sugar ring puckers are divided into 10 families described by the atom which is most displaced from the mean ring plane (C1′, C2′, C3′, C4′ or O4′) and the direction of this displacement (endo for displacements on the side of the C5′ atom and exo for displacements on the other side). These pucker families can be easily calculated from the phase angle and are also output by the Curves+ program.
In order to deal with non-standard nucleic acids the backbone parameters are not hard-wired into the program, but are contained in a data file (standard_s.lib) which can be modified or extended by this user. This makes it easy to analyse chemically modified backbones such as those, for example, in PNA (34 (link)).

Animals were euthanized with an overdose of sodium pentobarbital (100 mg/kg, intraperitoneally) after in vivo hemodynamic measurement, and then were rapidly perfused via inferior vena cava with precooled physiological saline for 5 min. The hearts were quickly removed, sectioned, and prepared for paraffin embedding and cryo-section. Routine staining techniques in paraffin embedded tissue (7-µm) included hematoxylin and eosin (H&E) staining (25˚C, 15 min) and Masson's trichrome staining for evaluating interstitial collagen deposition. Tetraethyl rhodamine isothiocyanate-conjugated wheat germ agglutinin (Invitrogen; Thermo Fisher Scientific, Inc.) plus 4,6-diamidino-2-phenylindole (DAPI; 5 mg/ml; Vector Laboratories, Inc.) staining was used to measure cardiomyocyte cross-sectional area, which was examined with a fluorescent microscope (80i; Nikon Corporation) as previously described by the authors (13 (link)). All image analysis was performed in a blinded manner by Image Pro Plus (version 4.5; Media Cybernetics, Inc.).
For detecting lipid droplet, Oil Red O staining was used in cryo-sectioned heart tissue (10 µm), and the sections were fixed with 10% buffered formalin (25˚C, 24 h) and stained with Oil Red O working solution (25˚C, 30 min). Hematoxylin was used for counterstaining. The perirenal adipose tissue was removed and smeared over a slide serving as the positive controls. For detecting sulfated proteoglycans, paraffin-embedded sections were stained with toluidine blue. Alcian blue/periodic acid-Schiff (AB-PAS) was used in paraffin-embedded sections to detect acidic sulfated mucins (AB positive), O-glycosides (PAS positive) and sialic acid (PAS positive) as previously described (14 (link)).
Transmission electron microscopy was performed for evaluating myocardial ultrastructure. Briefly, the samples from LV were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2; 4˚C, 24 h), post-fixed in 1.0% OsO4, dehydrated in alcohol and acetone solution, embedded in Epon812, sectioned with LKB ultramicrotome, and stained with uranyl acetate followed by lead citrate, then observed with a transmission electron microscope.