Structural investigation of a thermostable 1,2-ß-mannobiose phosphorylase from Thermoanaerobacter sp. X-514.

1,2-ß-Mannobiose phosphorylases (1,2-ß-MBPs) from glycoside hydrolase 130 (GH130) family are important bio-catalysts in glycochemistry applications owing to their ability in synthesizing oligomannans. Here, we report the crystal structure of a thermostable 1,2-ß-MBP from Thermoanaerobacter sp. X-514 termed Teth514_1789 to reveal the molecular basis of its higher thermostability and mechanism of action. We also solved the enzyme complexes of mannose, mannose-1-phosphate (M1P) and 1,4-ß-mannobiose to manifest the enzyme-substrate interaction networks of three main subsites. Notably, a Zn ion that should be derived from crystallization buffer was found in the active site and coordinates the phosphate moiety of M1P. Nonetheless, this Zn-coordination should reflect an inhibitory status as supplementing Zn severely impairs the enzyme activity. These results indicate that the effects of metal ions should be taken into consideration when applying Teth514_1789 and other related enzymes. Based on the structure, a reliable model of Teth514_1788 that shares 61.7% sequence identity to Teth514_1789 but displays a different substrate preference was built. Analyzing the structural features of these two closely related enzymes, we hypothesized that the length of a loop fragment that covers the entrance of the catalytic center might regulate the substrate selectivity. In conclusion, these information provide in-depth understanding of GH130 1,2-ß-MBPs and should serve as an important guidance for enzyme engineering for further applications.

Role of an e-Health Intervention in Holistic Healthcare: A Quasiexperiment in Patients Undergoing Cardiac Catheterization in Taiwan.

Background: The use of electronic health (e-health) resources is emerging as an alternative method to improve the secondary prevention of coronary artery disease (CAD). The aim of this study was to describe the influence of an e-health application in holistic healthcare for patients with CAD. Methods: A quasiexperiment with nonequivalent groups design recruited outpatients with a high risk of CAD admitted for cardiac catheterization. They were divided into two groups. Before the procedure, the control group received traditional patient education, and the intervention group watched videos on Internet-based social media. EQ-5D and FACIT-Sp-12 questionnaires were used as outcome measures of interest, and they were administered before and after the procedure and at the first return visit to the outpatient clinic after discharge. The effect of each intervention was tested using a linear mixed effects model. In addition, the 90-day readmission rate was also studied. Results: A total of 300 patients were divided into intervention and control groups (150 patients in each group). The interaction effect of EQ-5D was not statistically significant; however, improvements in FACIT-Sp-12 were greater in the intervention group from baseline to before discharge (regression coefficient (B) = 1.70, p < 0.001) and from baseline to postdischarge first outpatient visit (B = 1.81, p < 0.001). Moreover, the 90-day readmission rate was significantly lower in the intervention group (14% vs. 18.7%; p=0.016, log-rank test). Conclusions: e-health intervention with easily accessible Internet-based social media is a promising model to meet the holistic needs of patients with CAD in the modern era.

Squalene synthase as a target for Chagas disease therapeutics.

Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease.

Direct monitoring of chemical transformations by combining thin layer chromatography with nanoparticle-assisted laser desorption/ionization mass spectrometry.

A nanomaterial-assisted method that combines thin layer chromatography (TLC) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) was developed to directly monitor chemical transformations. A substrate-dependent extraction strategy was studied and successfully used to identify target molecules from the depths of a developed TLC plate. By using this strategy, a hydrophobic sample of interest was enriched on the surface of the TLC plate in the presence of acetonitrile, in contrast to using water and methanol to identify hydrophilic samples. The successful enrichment of samples by specific solvents provided stable desorption/ionization efficiencies of compounds of interest and led to very good sensitivity near the attomole scale. The method was then used to monitor 4-dimethylaminopyridine (DMAP)-catalyzed acylation in preparation of bifunctional sulfonamides. The labile DMAP-acyl intermediate and final sulfonamide product were clearly identified on TLC plates without external purification or sample preparation. Furthermore, in combination with collision-induced dissociation (CID) to provide structural information, the technique was successfully used in the natural product discovery of anti-inflammatory flavonoids from Helminthostachys zeylanica, a traditional Chinese herb. The newly proposed method provides a very low background from silica supports or organic matrices in the low molecular weight range (100-1000 Da). The technique may greatly accelerate studies of metabolomics, drug discovery, and organic synthesis.

Influence of diabetes on homocysteine-lowering therapy in chronic hemodialysis patients.

INTRODUCTION: The effect of homocysteine (Hcy)-lowering therapy may be different in hemodialysis (HD) patients with and without diabetes mellitus (DM). METHODS: Stable HD patients with uremia were administered folic acid and vitamin B for 3 months. The impact of treatment was compared in patients with and without DM. RESULTS: A total of 61 patients (31 men and 30 women) aged 56 ± 13 y completed the study. Among these, 44 patients (72%) did not have DM and 17 (28%) had DM. At baseline, total Hcy and high-sensitivity C-reactive protein (hsCRP) levels were similar. After treatment, the levels of total Hcy and hsCRP were significantly decreased in the nondiabetic group (total Hcy level decreased from 33.63 ± 14.13 µmol/l to 18.94 ± 8.46 µmol/l, p<0.001; hsCRP level decreased from 0.58 mg/dl [range, 0.21-1.05 mg/dl] to 0.22 mg/dl [range, 0.11-0.53 mg/dl], p<0.001) but not in the diabetic group (total Hcy level decreased from 34.97 ± 17.12 µmol/l to 29.53 ± 11.36 µmol/l, p=0.057; hsCRP level decreased from 0.80 mg/dl [range, 0.24-1.47 mg/dl] to 0.49 mg/dl [range, 0.45-0.98 mg/dl], p=0.28). Serial monitoring of total Hcy level showed a more sustained effect of therapy on patients without DM. CONCLUSION: Folic acid and vitamin B administration significantly lower total Hcy and hsCRP levels in HD patients without DM but not in those with DM.

Using oily wastewater emulsified fuel in boiler: energy saving and reduction of air pollutant emissions.

The limited data for using emulsified oil have demonstrated its effectiveness in reducing flue gas pollutant emissions. The presence of a high concentration of toxic organic compounds in industrial wastewaters always presents significant problems. Therefore, this study was undertaken by using wastewater with COD of 9600 mg/L and total petroleum hydrocarbons-gasoline 440 mg/L for making an emulsified oil (wastewater content 20% with 0.1% surfactant) to evaluate the extent of reductions in both criteria pollutants and polycyclic aromatic hydrocarbons. For comparison, two other systems (heavy oil fuel and water-emulsified oil) were also conducted. The wastewater-emulsified oil fuel results in significant reductions in particulate matter (PM), NO(x), SO2, and CO as compared to heavy oil fuel and similar to those from water/oil emulsified fuel; for PM, it is better in wastewater-emulsified oil. The reductions of total PAH flue gas emissions are 38 and 30% for wastewater- and water-emulsified fuel, respectively; they are 63 and 44% for total BaP(eq), respectively. In addition to reducing flue gas pollutant emissions, the results also demonstrate that the use of wastewater-emulsified fuel in boiler operation provides several advantages: (1) safe disposal of industrial wastewater; and (2) energy savings of about 13%. Thus, wastewater/oil-emulsified fuel is highly suitable for use in boilers.

Effects of aging and dietary antler supplementation on the calcium-regulating hormones and bone status in ovariectomized SAMP8 mice.

This study was conducted to investigate the effects of aging and long-term dietary antler supplementation on the calcium-regulating hormones and bone status in ovariectomized (Ovx) SAMP8 mice. The female SAMP8 mice were divided into four groups (in each group n = 6), Ovx or sham operated at the age of 2 months, and fed with 0.2% antler containing diet or control diet from the age of 2.5 months. The samples were collected at the age of 3, 6, 9, 12, and 15 months, respectively, for physicochemical analyses, biochemical analyses, and the determination of hormones by radioimmunoassay. The results showed that plasma calcium (Ca) concentrations were maintained in a narrow range in all groups throughout the whole experimental period. With aging and/or ovariectomy, plasma parathyroid hormone (PTH) and 1,25-dihydroxycholecalciferol (1,25-(OH)2-D3) levels increased, and plasma phosphorus (P) and calcitonin (CT) levels decreased, and the femoral bone densities and Ca contents increased during the earlier stage, and then decreased gradually in all groups. Plasma PTH and 1,25-(OH)2-D3 levels in the Ovx mice were significantly higher than those in the intact mice, and plasma P concentrations, plasma CT levels, femoral bone densities, and femoral Ca contents in the Ovx mice were significantly lower than those in the intact mice. In addition, the decreases of plasma P levels, plasma CT levels, femoral bone densities, and femoral Ca contents, and the increases of plasma PTH levels were moderated by antler administration in both Ovx and intact mice. However, there was no effect of the dietary antler supplementation on the plasma 1,25-(OH)2-D3 levels in the female mice. It is concluded that prolonged dietary antler supplementation has important positive effects on bone loss with age and/ or ovarian function deficiency.

Unusual cause of hypokalemic paralysis in aged men: Sjögren syndrome.

Hypokalemic paralysis is a less recognized but reversible disorder in elderly patients. This report describes two elderly Chinese males (age 74 and 78 years) who had progressive muscle weakness and eventually paralysis. Physical examination showed symmetrical flaccid paralysis of extremities. Both had the major biochemical abnormality of profound hypokalemia (1.4 and 1.8 mmol/L) accompanied by high urine K+ excretion and hyperchloremic metabolic acidosis. A positive urine anion gap and alkaline urine pointed to the diagnosis of distal renal tubular acidosis. Large doses of potassium chloride supplementation were required to restore muscle strength. Pertinent investigations, including elevated titers of antinuclear antibody and rheumatoid factor, positive anti-Ro antibody, low serum C3 and C4 levels, and delayed saliva excretion on salivary scintigraphy suggested Sjögren syndrome. Despite the lack of sicca syndrome at the initial presentation, both had development of typical sicca syndrome and positive Schirmer test at the 5-month and 1-year follow-up, respectively. Potassium citrate supplement and prednisolone therapy completely corrected the hypokalemia and metabolic acidosis. Extraglandular involvement with distal renal tubular acidosis preceding the typical sicca syndrome may induce hypokalemic paralysis and unveil Sjögren syndrome in elderly males.

Effects of age on plasma levels of calcium-regulating hormones and bone status in male SAMP8 mice.

This study was undertaken to examine whether the plasma levels of calcium-regulating hormones and bone status alter with age in male senescence accelerated mice (SAM), SAMP8. Age-matched senescence-resistant mice, SAMR1, were used as controls. The blood and femur samples were collected at 2.5 months of age (M) and then monthly from 3 to 12 M for physicochemical analyses, biochemical analyses, and the determination of hormones by radioimmunoassay. With advancing age, the plasma calcitonin (CT) levels decreased progressively, and the plasma parathyroid hormone (PTH) and 1,25-dihydroxycholecalciferol (1,25(OH)2D3) levels increased in both SAMR1 and SAMP8. The plasma calcium concentrations were maintained within a narrow range throughout the experimental period, while the plasma phosphorus (P) concentrations decreased with age in both strains. In contrast to SAMR1, the curves of age-related changes in the plasma CT levels and P concentrations were lower, and those in the plasma PTH levels were higher in SAMP8. The femoral bone densities and calcium contents increased gradually with age from the beginning of the experiment and peaked at 6 M in both strains, then declined. Those peaks were lower in SAMP8 than in SAMR1. These results indicate that the male SAMP8 develops osteoporotic signs earlier than SAMR1, and is proved to be a satisfactory animal model for longitudinal studies related to osteoporosis for men.