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

Rapid determination of 103 common veterinary drug residues in milk and dairy products by ultra performance liquid chromatography tandem mass spectrometry

A multi-residue method has been developed for the identification and quantification of 103 common veterinary drug residues in milk and dairy Products. This method was based on QuEChERS with dispersive solid-phase where C18 sorbent and anhydrous sodium sulfate were used to sample purification. After evaporation and reconstitution, the samples were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry. The mean recovery results were all higher than 60% except ampicillin, pipemidic acid, enoxacin, and estriol, and the relative standard deviation was <20.0%. The limit of quantification ranged between 0.1 and 5 μg/kg for milk and between 0.5 and 25 μg/kg for milk powder. It was successfully used to detect residues of veterinary drug in real samples. This study proposes a simple and fast analytical method for monitoring multi-class veterinary drug residues to ensure food safety

Sociodemographic and obstetric characteristics of stillbirths in China: a census of nearly 4 million health facility births between 2012 and 2014

Background Very little is known about the burden and determinants of stillbirths in China. We used data from a national surveillance system for health facility births to compute a stillbirth rate representative of all facility births in China and to explore sociodemographic and obstetric factors associated with variation in the stillbirth rate. Methods We used data from China’s National Maternal Near Miss Surveillance System between Jan 1, 2012, and Dec 31, 2014, which covers 441 hospitals in 326 urban districts and rural counties. The surveillance aimed to enumerate all maternal deaths and near misses in health facilities, and collected data prospectively for all pregnant or post-partum women admitted to the obstetric department. We restricted the analysis to births of 28 or more completed weeks of gestation or 1000 g or heavier birthweight. We examined the strength of association between sociodemographic characteristics, gestational age, and obstetric complications and stillbirths using logistic regression, taking account of the sampling strategy and clustering of births within hospitals and in cases of more than one birth per woman. Findings There were 3 956 836 births and 37 855 stillbirths, giving a stillbirth rate of 8·8 per 1000 births (95% CI 8·8–8·9). The stillbirth rate was particularly high for women younger than 15 years of age (59·9 stillbirths per 1000 births), those who had not sought antenatal care (38·3 per 1000), the unmarried (32·5 per 1000), those with no education (26·9 per 1000), or those who had had four or more births (23·2 per 1000). A high proportion (29 319 [78·2%] of 37 514) of stillbirths occurred at gestational ages of younger than 37 weeks, and about two thirds (24 787 [66·1%] of 37 514) were in women without any maternal complication at the time of birth. Of babies born at normal gestations (37–41 weeks), maternal complications substantially increased the risk of stillbirth (odds ratio comparing antepartum or intrapartum complications with no complication 3·96 [95% CI 3·66–4·29]), but only a small proportion (1638 [4·4%] of 37 514) of stillbirths fell into this group. Interpretation Our analysis of nearly 4 million births in 441 health facilities in China suggests a stillbirth rate of 8·8 per 1000 births between 2012 and 2014. Stillbirths do not feature in the Chinese Government’s 5 year plans and most information systems do not include stillbirths. The Government need to start paying attention to stillbirths and invest strategically in antenatal care, particularly for the most disadvantaged women, including the very young, unmarried, and illiterate, and those at high parity

Child feeding and stunting prevalence in left-behind children: a descriptive analysis of data from a central and western Chinese population

Objectives: To examine the effect of parental rural-to-urban internal migration on nutritional status of left-behind children and how this is related to guardianship. Methods: We used UNICEF China’s maternal and child health survey data to investigate stunting prevalence and feeding practices in children left behind by rural-to-urban internal migrant parents. We also assessed the effects of primary guardianship which is related closely with parental migration. Results: Of 6,136 children aged 0-3 years, over one third was left behind by one or both parents. About 13% were left behind by mothers, leaving guardianship primarily to grandmothers. Left-behind status was not associated with stunting, yet children who were cared for primarily by their fathers had a 32% increase of stunting compared to children cared for by the mothers (adjusted odds ratio[aOR]=1.32;95% confidence interval=1.04-1.67). Children with migrant mothers were less likely to receive age-appropriate breastfeeding (aOR=0.04;0.02-0.10) and a minimum acceptable diet (aOR=0.56;0.39-0.79) compared with non-left-behind children. Conclusions: Guardian’s feeding behaviours varied, and was inappropriate for both children affected and not affected by parent’s rural-to-urban internal migration. Community-based infant and young child feeding counselling and support should be provided to all caregivers

Generation of Functional CLL-Specific Cord Blood CTL Using CD40-Ligated CLL APC

PMCID: PMC3526610This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

A juvenile mouse model of anti-N-methyl-D-aspartate receptor encephalitis by active immunization

IntroductionAnti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a common autoimmune encephalitis, and it is associated with psychosis, dyskinesia, and seizures. Anti-NMDAR encephalitis (NMDARE) in juveniles and adults presents different clinical charactreistics. However, the pathogenesis of juvenile anti-NMDAR encephalitis remains unclear, partly because of a lack of suitable animal models.MethodsWe developed a model of juvenile anti-NMDAR encephalitis using active immunization with an amino terminal domain peptide from the GluN1 subunit (GluN1356 − 385) against NMDARs in 3-week-old female C57BL/6J mice.ResultsImmunofluorescence staining suggested that autoantibody levels in the hippocampus increased, and HEK-293T cells staining identified the target of the autoantibodies as GluN1, suggesting that GluN1-specific immunoglobulin G was successfully induced. Behavior assessment showed that the mice suffered significant cognition impairment and sociability reduction, which is similar to what is observed in patients affected by anti-NMDAR encephalitis. The mice also exhibited impaired long-term potentiation in hippocampal CA1. Pilocarpine-induced epilepsy was more severe and had a longer duration, while no spontaneous seizures were observed.ConclusionThe juvenile mouse model for anti-NMDAR encephalitis is of great importance to investigate the pathological mechanism and therapeutic strategies for the disease, and could accelerate the study of autoimmune encephalitis

China's human resources for maternal and child health:a national sampling survey

Background: In order to achieve the Millennium Development Goals (MDG) 4 and 5, the Chinese Government has invested greatly in improving maternal and child health (MCH) with impressive results. However, one of the most important barriers for further improvement is the uneven distribution of MCH human resources. There is little information about the distribution, quantity and capacity of the Chinese MCH human resources and we sought to investigate this. Methods: Cities at prefectural level were selected by random cluster sampling. All medical and health institutions providing MCH-related services in the sampled areas were investigated using a structured questionnaire. The data were weighted based on the proportion of the sampled districts/cities. Amount, proportions and numbers per 10,000 population of MCH human resources were estimated in order to reveal the quantity of the Chinese MCH human resources. The capacity of MCH human resources was evaluated by analyzing data on the education level and professional skills of the staff. Results: There were 77,248 MCH workers in China in 2010. In general, 67.6 % and 71.9 % of the women's and children's health care professionals had an associate degree or higher, whereas around 30 % had only high-school or lower degrees. More than 40 % of the women's health workers were capable of providing skilled birth attendance, but these proportions varied between different institutions and locations. Conclusions: Evidence from this study highlights that Chinese MCH human resources are not in shortage in the national level. However, the quantity and capacity of MCH human resources are not evenly distributed among different institutions and locations. Finally there is a need in the improvement of the MCH services by improving the quality of MCH human resources.UNICEFSCI(E)[email protected]

TDRD5 Is Required for Spermatogenesis and Oogenesis in Locusta migratoria

Tudor family proteins exist in all eukaryotic organisms and play a role in many cellular processes by recognizing and binding to proteins with methylated arginine or lysine residues. TDRD5, a member of Tudor domain-containing proteins (TDRDs), has been implicated in the P-element-induced wimpy testis-interacting RNA (piRNA) pathway and germ cell development in some model species, but little is known about its function in other species. Therefore, we identified and characterized LmTDRD5, the TDRD5 ortholog in Locusta migratoria, a hemimetabolous pest. The LmTdrd5 gene has 19 exons that encode a protein possessing a single copy of the Tudor domain and three LOTUS domains at its N-terminus. qRT-PCR analysis revealed a high LmTdrd5 expression level in genital glands. Using RNA interference, LmTdrd5 knockdown in males led to a lag in meiosis phase transition, decreased spermatid elongation and sperm production, and downregulated the expression of the two germ cell-specific transcription factors, LmCREM and LmACT, as well as the sperm tail marker gene LmQrich2.LmTdrd5 knockdown in females reduced the expression levels of vitellogenin (Vg) and Vg receptor (VgR) and impaired ovarian development and oocyte maturation, thus decreasing the hatchability rate. These results demonstrate that LmTdrd5 is essential for germ cell development and fertility in locusts, indicating a conserved function for TDRD5