Evaluation of method performance for oxidative stress biomarkers in urine and biological variations in urine of patients with type 2 diabetes mellitus and diabetic nephropathy

Background

Oxidative stress biomarkers such as superoxide dismutase (CuZnSOD), catalase (CAT) and malondialdehyde (MDA) play an important role in the pathogenesis or progression of numerous diseases. Data regarding the biological variation and analytical quality specifications (imprecision, bias and total error) for judging the acceptability of method performance for oxidative stress biomarkers in urine are conspicuously lacking in the literature. Such data are important in setting analytical quality specifications, assessing the utility of population reference intervals (index of individuality) and assessing the significance of changes in serial results from an individual (reference change value; RCV).

Materials and methods

20 patients with type 2 diabetes mellitus (T2DM), 20 patients with diabetic nephropathy (DN) and 14 healthy individuals as control were involved in this study. Timed first morning urine samples were taken from patients and healthy groups on the zero, 1st, 3rd, 5th, 7th, 15th and 30th days. Index of individuality and reference change value were calculated from within-subject and between-subject variations. Methods of oxidative stress biomarkers in human blood were adopted in human urine and markers were measured as spectrophotometrically. Also, analytical quality specifications for evaluation of the method performance were established for oxidative stress biomarkers in urine.

Results

Within-subject variations of oxidative stress biomarkers were significantly higher in patients with DN and T2DM compared to healthy subjects. MDA showed low individuality, and within-subject variances of MDA were larger than between-subject variances in all groups. However, CAT and CuZnSOD showed strong individuality, but within-subject variances of them were smaller than between-subject variances in all groups. RCVs of all analytes in diabetic patients were relatively higher, because of high within-subject variation, resulting in a higher RCV. Also, the described methodology achieves these goals, with analytical CVs of < 3.5% for all analytes. Goals for bias and total error were 6.0-7.9% and 12.5-23.3%, respectively.

Conclusions

RCVs concept for predicting the clinical status in diabetic patients represents an optimization of laboratory reporting and could be a valuable tool for clinical decision. Furthermore, for oxidative stress biomarkers’ measurements in urine, the desirable imprecision goals based on biological variation are obtainable by current methodologies.     

Optimization of NMR spectroscopy of encapsulated proteins dissolved in low viscosity fluids

Comprehensive application of solution NMR spectroscopy to studies of macromolecules remains fundamentally limited by the molecular rotational correlation time. For proteins, molecules larger than 30 kDa require complex experimental methods, such as TROSY in conjunction with isotopic labeling schemes that are often expensive and generally reduce the potential information available. We have developed the reverse micelle encapsulation strategy as an alternative approach. Encapsulation of proteins within the protective nano-scale water pool of a reverse micelle dissolved in ultra-low viscosity nonpolar solvents overcomes the slow tumbling problem presented by large proteins. Here, we characterize the contributions from the various components of the protein-containing reverse micelle system to the rotational correlation time of the encapsulated protein. Importantly, we demonstrate that the protein encapsulated in the reverse micelle maintains a hydration shell comparable in size to that seen in bulk solution. Using moderate pressures, encapsulation in ultra-low viscosity propane or ethane can be used to magnify this advantage. We show that encapsulation in liquid ethane can be used to reduce the tumbling time of the 43 kDa maltose binding protein from ~23 to ~10 ns. These conditions enable, for example, acquisition of TOCSY-type data resolved on the adjacent amide NH for the 43 kDa encapsulated maltose binding protein dissolved in liquid ethane, which is typically impossible for proteins of such size without use of extensive deuteration or the TROSY effect.     

Structural cooperativity in histone H3 tail modifications

Post-translational modifications of histone H3 tails have crucial roles in regulation of cellular processes. There is cross-regulation between the modifications of K4, K9, and K14 residues. The modifications on these residues drastically promote or inhibit each other. In this work, we studied the structural changes of the histone H3 tail originating from the three most important modifications; tri-methylation of K4 and K9, and acetylation of K14. We performed extensive molecular dynamics simulations of four types of H3 tails: (i) the unmodified H3 tail having no chemical modification on the residues, (ii) the tri-methylated lysine 4 and lysine 9 H3 tail (K4me3K9me3), (iii) the tri-methylated lysine 4 and acetylated lysine 14 H3 tail (K4me3K14ace), and (iv) tri-methylated lysine 9 and acetylated lysine 14 H3 tail (K9me3K14ace). Here, we report the effects of K4, K9, and K14 modifications on the backbone torsion angles and relate these changes to the recognition and binding of histone modifying enzymes. According to the Ramachandran plot analysis; (i) the dihedral angles of K4 residue are significantly affected by the addition of three methyl groups on this residue regardless of the second modification, (ii) the dihedral angle values of K9 residue are similarly altered majorly by the tri-methylation of K4 residue, (iii) different combinations of modifications (tri-methylation of K4 and K9, and acetylation of K14) have different influences on phi and psi values of K14 residue. Finally, we discuss the consequences of these results on the binding modes and specificity of the histone modifying enzymes such as DIM-5, GCN5, and JMJD2A.     

Rheological Behaviour of Instant Hot Chocolate Beverage: Part 1. Optimization of the Effect of Different Starches and Gums

In this study, effects of different starches (tapioca (TS), wheat (WS), corn (CS), potato (PS), modified corn (MCS) and modified potato (MPS)) and gums (xanthan gum (XG), guar gum (GG), alginate (A), salep (S), locust bean gum (LBG) and carrageen (C)) on the rheological properties of model hot chocolate beverage were studied. Swelling power (SP) of the starches and water absorption capacity (WAC) of the gums were determined. Hot chocolate beverages showed pseudoplastic behaviour. Ostwald de Waele model accurately described flow behaviour of each beverage sample. K, n, R ² values for Ostwald model were in the range of 4.8–160.3 mPa.sⁿ, 0.5117–0.9745, 0.9972–0.9998, respectively. The highest synergic effect in the model was observed between the interaction of MCS and XG. The XG-PS, XG-TS, XG-CS combinations showed the highest K and viscosity values, respectively.     

Serratia ficaria isolated from sputum specimen

Serratia ficaria was first described in 1979 as a Gram-negative facultative anaerobic rod. S. ficaria was found in figs, but also isolated from human specimens in a few cases. We now report an isolate of S. ficaria from sputum specimen.A 46-year-old man was suffering from a chronic renal failure of five years, four months of peritoneal dialysis and one week of fever due to respiratory tract infection, accompanied by cough. Sputum culture yielded a Gram-negative rod. It was identified as S. ficaria and the antibiotic susceptibility test was performed by automated Vitek II (bioMerieux). The tested S. ficaria strain was susceptible to amikacin, gentamicin, cefepime, trimethoprim-sulfamethoxazole, imipenem, meropenem, tigecycline and ciprofloxacin. This strain was resistant to ampicillin, amoxicillin-clavulanic acid, cephalothin, cefoxitine, cefuroxime and ceftriaxone. The patient was treated successfully (80 mg trimethoprim/400 mg sulfamethoxazole twice daily for 7 days)S. ficaria is an opportunistic pathogen responsible for intestinal colonization or serious infections such as septicaemia, gall bladder empyema in immunocompromised patients. The fig tree and fig play an important role in human colonization. It should be remembered that S. ficaria infections may be encountered frequently especially in fig tree culture zones.