An improved approach for extraction and high-performance liquid chromatography analysis of paraquat in human plasma

A simple, sensitive, reliable and economical HPLC method for quantifying paraquat concentration in human plasma has been developed, using diethyl paraquat as an internal standard. The drugs were extracted from the sample and separated on Xtimate C18 column with a mobile phase of 15% acetonitrile in 0.1M orthophosphoric acid containing SDS (150 mg/l). The pH of the mobile phase was adjusted to 3 with triethylamine and the detection wavelength was 256 nm for both paraquat and the internal standard. The average extraction recoveries were 91.9%. Good linearity (R(2)=0.9984) was observed throughout the range of 0.02-10 μg/ml in 0.5 ml plasma. The overall accuracy of this method was 97.6-107.3% and the lower limit of detection was 0.01 μg/ml. The intra- and inter-day variations were lower than 3.65% and 2.64%, respectively. We used this method to examine the paraquat concentrations of 53 patients with acute paraquat intoxication of whom 26 (49.1%) survived. In conclusion, this method was suitable for quantification of paraquat plasma concentration in toxicological samples. It was helpful in both assessing the severity of intoxication and predicting the outcome of paraquat poisoning.     

Molecular dynamics simulation to investigate the impact of disulfide bond formation on conformational stability of chicken cystatin I66Q mutant

Chicken cystatin (cC) mutant I66Q is located in the hydrophobic core of the protein and increases the propensity for amyloid formation. Here, we demonstrate that under physiological conditions, the replacement of Ile with the Gln in the I66Q mutant increases the susceptibility for the disulfide bond Cys71–Cys81 to be reduced when compared to the wild type (WT) cC. Molecular dynamics (MD) simulations under conditions favoring cC amyloid fibril formation are in agreement with the experimental results. MD simulations were also performed to investigate the impact of disrupting the Cys71–Cys81 disulfide bond on the conformational stability of cC at the atomic level, and highlighted major disruption to the cC appendant structure. Domain swapping and extensive unfolding has been proposed as one of the possible mechanisms initiating amyloid fibril formation by cystatin. Our in silico studies suggest that disulfide bond formation between residues Cys95 and Cys115 is necessary to maintain conformational stability of the I66Q mutant following breakage of the Cys71–Cys81 disulfide bridge. Subsequent breakage of disulfide bond Cys95–Cys115 resulted in large structural destabilization of the I66Q mutant, which increased the α–β interface distance and expanded the hydrophobic core. These experimental and computational studies provide molecular-level insight into the relationship between disulfide bond formation and progressive unfolding of amyloidogenic cC mutant I66Q.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:23     

The Relationship between Endogenous Androgens and Body Fat Distribution in Early and Late Postmenopausal Women

Objectives

To investigate the relationship between endogenous androgens and body fat distribution in early and late postmenopausal women.

Materials and Methods

We enrolled postmenopausal women consisting of an early group (≤5 years since menopause, n = 105) and a late group (≥10 years since menopause, n = 107). Each group was subdivided into normal weight (BMI <24 kg/m²) group, overweight and obese (BMI ≥24 kg/m²) group. Fasting total testosterone (T), dehydroepiandrosterone-sulfate (DHEA-S) and sex hormone-binding globulin (SHBG) levels were measured. Body fat distribution was evaluated by dual-energy X-ray absorptiometry (DEXA).

Results

Late postmenopausal women had a higher proportion of body fat than early postmenopausal women. The body fat of the overweight and obese women had a greater tendency to accumulate in the abdomen compared with the normal weight women both in early and late postmenopausal groups. The overweight and obese women had a higher free testosterone (FT) than the normal weight women in early postmenopausal women (P<0.05). In late postmenopausal women, the overweight and obese women had higher DHEA-S levels than normal weight women (P<0.05). No direct relationship was observed between the T levels and body fat distribution both in early and late postmenopausal groups (P>0.05).The FT in early postmenopausal women and the DHEA-S levels in late postmenopausal women correlated positively with the trunk/leg fat ratio (T/L) and the proportion of android fat whereas correlated negatively with the proportion of gynoid fat in the partial correlation and multiple linear regression analyses (all P<0.05).

Conclusions

Serum T levels do not correlate directly with body fat distribution, the FT in early postmenopausal women and DHEA-S levels in late postmenopausal women correlate positively with abdominal fat accumulation.     

Molecular imprinting of nitrophenol and hydroxybenzoic acid isomers: effect of molecular structure and acidity on imprinting

Three nitrophenol isomer-imprinted polymers were prepared under the same conditions using 4-vinylpyridine as a functional monomer. Different recognition capacities for template molecules were observed for the three polymers. Another imprinting system with stronger acidity than nitrophenol isomers, 2-hydroxybenzoic acid (salicylic acid) and 4-hydroxybenzoic acid, was imprinted using 4-vinylpyridine or acrylamide as functional monomer respectively. Both 4-hydroxybenzoic acid-imprinted polymers using the two monomers showed recognition ability for the template molecule. However, when acrylamide was chosen as functional monomer, the salicylic acid-imprinted polymer showed very weak recognition for the template molecule, whereas strong recognition ability of the resultant polymer for salicylic acid was observed with 4-vinylpyridine as functional monomer. It seems that the structure and acidity of template molecules is responsible for the difference in recognition, by influencing the formation and strength of interaction between template molecule and functional monomer during the imprinting process. An understanding of the mechanism of molecular imprinting and molecular recognition of MIPs will help to predict the selectivity of MIPs on the basis of template molecule properties.     

韭菜线粒体锰超氧化物歧化酶纯化及性质研究

经硫酸铵沉淀、ＤＥＡＥ－Ｓｅｐｈａｃｅｌ层析和Ｓｅｐｈａｄｅｘ Ｇ－２００凝胶过滤,将韭菜线粒体ＳＯＤ纯化到均一程度。从６０００ｇ韭菜叶片线粒体中纯化得到２．５ｍｇ酶,酶比活力达１２００Ｕ／ｍｇ蛋白。该酶对ＫＣＮ和Ｈ２Ｏ２都不敏感,热稳定性弱 外光区吸收峰在２８０ｎｍ,凝胶过滤法测得其分子量为８２００Ｄ,ＳＯＳ－ＰＡＧＥ法测定其亚基分子量的２２０００Ｄ,ＤＮＳ法测得其Ｎ－末端氨基酸为缬氨酸。上述结     

Investigation of Repeat Client Drop-Out and Re-Enrolment Cycles in Fourteen Methadone Maintenance Treatment Clinics in Guangdong,China

ObjectiveClient adherence is vital for effective methadone maintenance treatment (MMT). This study explores the pattern and associated factors of client adherence, drop-out and re-enrolment in the Chinese MMT programme over the period of 2006–2013.MethodsThis retrospective study was conducted in 14 MMT clinics in Guangdong Province, China. We employed Kaplan-Meier survival analysis to estimate the rates of drop-out and re-enrolment of MMT clients and multivariate Cox regression to identify associated factors.ResultsAmong 1,512 study participants, 79% have experienced ‘drop-out’ during the 7-year study period. However, 82% ‘dropped-out’ clients resumed treatment at a later time. Low education level (junior high or below versus otherwise, HR = 1.21, 1.05–1.40), low methadone dosage in the first treatment episode (<50 ml versus ≥50 ml, HR = 1.84, 1.64–2.06) and higher proportion of positive urine test (≥50% versus<50%, HR = 3.72, 3.30–4.20) during the first treatment episode were strong predictors of subsequent drop-outs of the participants. Among the ‘dropped-out’ clients, being female (HR = 1.40, 1.23–1.60), being married (HR = 1.19, 1.09–1.30), and having a higher proportion of positive urine tests in the first treatment episode (≥50% versus<50%, HR = 1.35, 1.20–1.51) had greater likelihood of subsequent re-enrolment in MMT. Clients receiving lower methadone dosage (first treatment episode <50 ml versus ≥50 ml, HR = 1.12, 1.03–1.23; the last intake before drop-out <50 ml versus ≥50 ml, HR = 1.16, 1.04–1.30) were also more likely to re-enrol.ConclusionPersistent cycling in-and-out of clients in MMT programmes is common. Insufficient dosage and higher proportion of positive urine samples in the first treatment episode are the key determinants for subsequent client drop-out and re-enrolment. Interventions should target clients in their early stage of treatment to improve retention in the long term.     

Rural-to-Urban Migrants' Experiences with Primary Care under Different Types of Medical Institutions in Guangzhou,China

Objectives

China is facing the unprecedented challenge of rapidly increasing rural-to-urban migration. Migrants are in a vulnerable state when they attempt to access to primary care services. This study was designed to explore rural-to-urban migrants’ experiences in primary care, comparing their quality of primary care experiences under different types of medical institutions in Guangzhou, China.

Methods

The study employed a cross-sectional survey of 736 rural-to-urban migrants in Guangzhou, China in 2014. A validated Chinese version of Primary Care Assessment Tool—Adult Short Version (PCAT-AS), representing 10 primary care domains was used to collect information on migrants’ quality of primary care experiences. These domains include first contact (utilization), first contact (accessibility), ongoing care, coordination (referrals), coordination (information systems), comprehensiveness (services available), comprehensiveness (services provided), family-centeredness, community orientation and culturally competent. These measures were used to assess the quality of primary care performance as reported from patients’ perspective. Analysis of covariance was conducted for comparison on PCAT scores among migrants accessing primary care in tertiary hospitals, municipal hospitals, community health centers/community health stations, and township health centers/rural health stations. Multiple linear regression models were used to explore factors associated with PCAT total scores.

Results

After adjustments were made, migrants accessing primary care in tertiary hospitals (25.49) reported the highest PCAT total scores, followed by municipal hospitals (25.02), community health centers/community health stations (24.24), and township health centers/rural health stations (24.18). Tertiary hospital users reported significantly better performance in first contact (utilization), first contact (accessibility), coordination (information system), comprehensiveness (service available), and cultural competence. Community health center/community health station users reported significantly better experience in the community orientation domain. Township health center/rural health station users expressed significantly better experience in the ongoing care domain. There were no statistically significant differences across settings in the ongoing care, comprehensiveness (services provided), and family-centeredness domains. Multiple linear regression models showed that factors positively associated with higher PCAT total scores also included insurance covering parts of healthcare payment (P<0.001).

Conclusions

This study highlights the need for improvement in primary care provided by primary care institutions for rural-to-urban migrants. Relevant policies related to medical insurance should be implemented for providing affordable healthcare services for migrants accessing primary care.     

A Novel Peroxidase from Fresh Fruiting Bodies of the Mushroom Pleurotus pulmonarius

International Journal of Peptide Research and Therapeutics - A novel peroxidase has been isolated from fresh fruiting bodies of the mushroom Pleurotus pulmonarius, with a purification protocol of...     

Microbial production of amino acid-modified spider dragline silk protein with intensively improved mechanical properties

Spider dragline silk is a remarkably strong fiber with impressive mechanical properties, which were thought to result from the specific structures of the underlying proteins and their molecular size. In this study, silk protein 11R26 from the dragline silk protein of Nephila clavipes was used to analyze the potential effects of the special amino acids on the function of 11R26. Three protein derivatives, ZF4, ZF5, and ZF6, were obtained by site-directed mutagenesis, based on the sequence of 11R26, and among these derivatives, serine was replaced with cysteine, isoleucine, and arginine, respectively. After these were expressed and purified, the mechanical performance of the fibers derived from the four proteins was tested. Both hardness and average elastic modulus of ZF4 fiber increased 2.2 times compared with those of 11R26. The number of disulfide bonds in ZF4 protein was 4.67 times that of 11R26, which implied that disulfide bonds outside the poly-Ala region affect the mechanical properties of spider silk more efficiently. The results indicated that the mechanical performances of spider silk proteins with small molecular size can be enhanced by modification of the amino acids residues. Our research not only has shown the feasibility of large-scale production of spider silk proteins but also provides valuable information for protein rational design.