Solid-Phase Extraction Combined with High Performance Liquid Chromatography-Diode Array Detector for Rapid Determination of Estrogens in Milk

Purpose: To develop a simple but effective method for the quantitative analysis of estrogens in milk.Methods: Solid-phase extraction method was employed for the extraction of the estrogen from milk and high performance liquid chromatography-diode array detector (HPLC-DAD) was used for the determination of estrogen.Results: Optimal chromatographic conditions were achieved on an Eclipse XDB-C18 column at a flow rate of 0.8 mL/min at room temperature. The products were monitored using a DAD detector set at 280 nm. The retention time of the three main estrogens were 15 min with a gradient program of acetonitrile/water, from 0 to 3 min, holding 35 % acetonitrile; from 4 to 7 min, ramped to 55 % acetonitrile; from 8 to 20 min, maintained at 55 % acetonitrile. The results showed that the calibration curve had good linearity within the concentration range of 0.5 - 8.0 μg/mL with correlation coefficient (R2) in the range of 0.9929-0.9936. The limit of detection was in the range of 0.025 - 0.045 μg/mL while mean recovery of estrogen from milk samples varied from 92.6 to 104.1 %. Satisfactory precision was obtained both for intra-assay (RSD, 1.8 to 4.4 %) and inter-assay (RSD, 2.0 to 4.7 %).Conclusion: The proposed method is environmentally friendly, inexpensive and convenient, and should be helpful in analyzing estrogens in biological, environmental and food samples.Keywords: Solid-phase extraction, Milk, Estrogens, High performance liquid chromatography-diode array detecto

Isolation, purification and PEG-mediated transient expression of mesophyll protoplasts in Camellia oleifera

Background: Camellia oleifera (C. oleifera) is a woody edible oil crop of great economic importance. Because of the lack of modern biotechnology research, C. oleifera faces huge challenges in both breeding and basic research. The protoplast and transient transformation system plays an important role in biological breeding, plant regeneration and somatic cell fusion. The objective of this present study was to develop a highly efficient protocol for isolating and purifying mesophyll protoplasts and transient transformation of C. oleifera. Several critical factors for mesophyll protoplast isolation from C. oleifera, including starting material (leaf age), pretreatment, enzymatic treatment (type of enzyme, concentration and digestion time), osmotic pressure and purification were optimized. Then the factors affecting the transient transformation rate of mesophyll protoplasts such as PEG molecular weights, PEG4000 concentration, plasmid concentration and incubation time were explored.Results: The in vitro grown seedlings of C. oleifera 'Huashuo' were treated in the dark for 24 h, then the 1st to 2nd true leaves were picked and vacuumed at - 0.07 MPa for 20 min. The maximum yield (3.5 x 10(7)/g.W) and viability (90.9%) of protoplast were reached when the 1st to 2nd true leaves were digested in the enzymatic solution containing1.5% (w/v) Cellulase R-10, 0.5% (w/v) Macerozyme R-10 and 0.25% (w/v) Snailase and 0.4 M mannitol for 10 h. Moreover, the protoplast isolation method was also applicable to the other two cultivars, the protoplast yield for 'TXP14' and 'DP47' was 1.1 x 10(7)/g.FW and 2.6 x 10(7)/g. FW, the protoplast viability for 'TXP14' and 'DP47' was 90.0% and 88.2%. The purification effect was the best when using W buffer as a cleaning agent by centrifugal precipitation. The maximum transfection efficiency (70.6%) was obtained with the incubation of the protoplasts with 15 mu g plasmid and 40% PEG4000 for 20 min.Conclusion: In summary, a simple and efficient system for isolation and transient transformation of C. oleifera mesophyll protoplast is proposed, which is of great significance in various aspects of C. oleifera research, including the study of somatic cell fusion, genome editing, protein function, signal transduction, transcriptional regulation and multi-omics analyses

Proteome quantification of cotton xylem sap suggests the mechanisms of potassium-deficiency-induced changes in plant resistance to environmental stresses

Proteomics was employed to investigate the molecular mechanisms of apoplastic response to potassium(K)-deficiency in cotton. Low K (LK) treatment significantly decreased the K and protein contents of xylem sap. Totally, 258 peptides were qualitatively identified in the xylem sap of cotton seedlings, of which, 90.31% were secreted proteins. Compared to the normal K (NK), LK significantly decreased the expression of most environmental-stress-related proteins and resulted in a lack of protein isoforms in the characterized proteins. For example, the contents of 21 Class Ш peroxidase isoforms under the LK were 6 to 44% of those under the NK and 11 its isoforms were lacking under the LK treatment; the contents of 3 chitinase isoforms under LK were 11–27% of those under the NK and 2 its isoforms were absent under LK. In addition, stress signaling and recognizing proteins were significantly down-regulated or disappeared under the LK. In contrast, the LK resulted in at least 2-fold increases of only one peroxidase, one protease inhibitor, one non-specific lipid-transfer protein and histone H4 and in the appearance of H2A. Therefore, K deficiency decreased plant tolerance to environmental stresses, probably due to the significant and pronounced decrease or disappearance of a myriad of stress-related proteins

Relaxation of the one child policy and trends in caesarean section rates and birth outcomes in China between 2012 and 2016: observational study of nearly seven million health facility births.

OBJECTIVE: To examine how the relaxation of the one child policy and policies to reduce caesarean section rates might have affected trends over time in caesarean section rates and perinatal and pregnancy related mortality in China. DESIGN: Observational study. SETTING: China's National Maternal Near Miss Surveillance System (NMNMSS). PARTICIPANTS: 6 838 582 births at 28 completed weeks or more of gestation or birth weight ≥1000 g in 438 hospitals in the NMNMSS between 2012 and 2016. MAIN OUTCOME MEASURES: Obstetric risk was defined using a modified Robson classification. The main outcome measures were changes in parity and age distributions and relative frequency of each Robson group, crude and adjusted trends over time in caesarean section rates within each risk category (using Poisson regression with a robust variance estimator), and trends in perinatal and pregnancy related mortality over time. RESULTS: Caesarean section rates declined steadily between 2012 and 2016 (crude relative risk 0.91, 95% confidence interval 0.89 to 0.93), reaching an overall hospital based rate of 41.1% in 2016. The relaxation of the one child policy was associated with an increase in the proportion of multiparous births (from 34.1% in 2012 to 46.7% in 2016), and births in women with a uterine scar nearly doubled (from 9.8% to 17.7% of all births). Taking account of these changes, the decline in caesarean sections was amplified over time (adjusted relative risk 0.82, 95% confidence interval 0.81 to 0.84). Caesarean sections declined noticeably in nulliparous women (0.75, 0.73 to 0.77) but also declined in multiparous women without a uterine scar (0.65, 0.62 to 0.77). The decrease in caesarean section rates was most pronounced in hospitals with the highest rates in 2012, consistent with the government's policy of targeting hospitals with the highest rates. Perinatal mortality declined from 10.1 to 7.2 per 1000 births over the same period (0.87, 0.83 to 0.91), and there was no change in pregnancy related mortality over time. CONCLUSIONS: China is the only country that has succeeded in reverting the rising trends in caesarean sections. China's success is remarkable given that the changes in obstetric risk associated with the relaxation of the one child policy would have led to an increase in the need for caesarean sections. China's experience suggests that change is possible when strategies are comprehensive and deal with the system level factors that underpin overuse as well as the various incentives at work during a clinical encounter