Overexpression of NtGCN2 improves drought tolerance in tobacco by regulating proline accumulation, ROS scavenging ability, and stomatal closure.

Drought stress is a severe threat to plants. Genes that respond to drought stress are essential for plant growth and development. General control nonderepressible 2 (GCN2) encodes a protein kinase that responds to various biotic and abiotic stresses. However, the mechanism of GCN2 in plant drought tolerance remains unclear. In the present study, the promoters of NtGCN2 from Nicotiana tabacum K326, which contained a drought-responsive Cis-acting element MYB that can be activated by drought stress, were cloned. Furthermore, the drought tolerance function of NtGCN2 was investigated using NtGCN2-overexpressed transgenic tobacco plants. NtGCN2-overexpressed transgenic plants were more tolerant to drought stress than wild-type (WT) plants. The transgenic tobacco plants exhibited higher proline and abscisic acid (ABA) contents, antioxidant enzyme activities, leaf relative water content, and expression levels of genes encoding key antioxidant enzymes and proline synthase, but lower levels of malondialdehyde and reactive oxygen species, and reduced stomatal apertures, stomatal densities, and stomatal opening rates compared to WT plants under drought stress. These results indicated that overexpression of NtGCN2 conferred drought tolerance in transgenic tobacco plants. RNA-seq analysis showed that overexpression of NtGCN2 responded to drought stress by regulating the expression of genes related to proline synthesis and catabolism, abscisic acid synthesis and catabolism, antioxidant enzymes, and ion channels in guard cells. These results showed that NtGCN2 might regulate drought tolerance by regulating proline accumulation, reactive oxygen species (ROS) scavenging, and stomatal closure in tobacco and may have the potential for application in the genetic modification of crop drought tolerance.

A new feature selection approach for driving fatigue EEG detection with a modified machine learning algorithm.

This study aims to identify new electroencephalography (EEG) features for the detection of driving fatigue. The most common EEG feature in driving fatigue detection is the power spectral density (PSD) of five frequency bands, i.e., alpha, beta, gamma, delta, and theta bands. PSD has proved to be useful, however its flaw is that it covers much implicit information of the time domain. In this study we propose a new approach, which combines ensemble empirical mode decomposition (EEMD) and PSD, to explore new EEG features for driving fatigue detection. Through EEMD we get a series of intrinsic mode function (IMF) components, from which we can extract PSD features. We used six features to compare with the proposed features, including the PSD of five frequency bands, PSD of empirical mode decomposition (EMD)-IMF components, PSD, permutation entropy (PE), sample entropy (SE), and fuzzy entropy (FE) of EEMD-IMF components, and common spatial pattern. Feature overlap ratio and multiple machine learning methods were applied to evaluate these feature extraction approaches. The results show that the classification accuracy and overlap ratio of experiments based on IMF's energy spectrum is far superior to other features. Through channel optimization and a comparison of accuracy, we conclude that our new feature selection approach has a better performance based on the modified hierarchical extreme learning machine algorithm with Particle Swarm Optimization (PSO-H-ELM) classifier, which has the highest average accuracy of 97.53%.

3D-QSAR, molecular docking, and molecular dynamics simulation study of thieno[3,2-b]pyrrole-5-carboxamide derivatives as LSD1 inhibitors.

Histone Lysine Specific Demethylase 1 (LSD1) is overexpressed in many cancers and becomes a new target for anticancer drugs. In recent years, small molecule inhibitors with various structures targeting LSD1 have been reported. Here we report the binding interaction modes of a series of thieno[3,2-b]pyrrole-5-carboxamide LSD1 inhibitors using molecular docking, and three-dimensional quantitative structure-activity relationships (3D-QSAR). Comparative molecular field analysis (CoMFA q 2 = 0.783, r 2 = 0.944, r pred 2 = 0.851) and comparative molecular similarity indices analysis (CoMSIA q 2 = 0.728, r 2 = 0.982, r pred 2 = 0.814) were used to establish 3D-QSAR models, which had good verification and prediction capabilities. Based on the contour maps and the information of molecular docking, 8 novel small molecules were designed in silico, among which compounds D4, D5 and D8 with high predictive activity were subjected to further molecular dynamics simulations (MD), and their possible binding modes were explored. It was found that Asn535 plays a crucial role in stabilizing the inhibitors. Furthermore, ADME and bioavailability prediction for D4, D5 and D8 were carried out. The results would provide valuable guidance for designing new reversible LSD1 inhibitors in the future.

Investigating the Binding Mode of Reversible LSD1 Inhibitors Derived from Stilbene Derivatives by 3D-QSAR, Molecular Docking, and Molecular Dynamics Simulation.

Overexpression of lysine specific demethylase 1 (LSD1) has been found in many cancers. New anticancer drugs targeting LSD1 have been designed. The research on irreversible LSD1 inhibitors has entered the clinical stage, while the research on reversible LSD1 inhibitors has progressed slowly so far. In this study, 41 stilbene derivatives were studied as reversible inhibitors by three-dimensional quantitative structure-activity relationship (3D-QSAR). Comparative molecular field analysis (CoMFA q 2 = 0.623, r 2 = 0.987, r pred 2 = 0.857) and comparative molecular similarity indices analysis (CoMSIA q 2 = 0.728, r 2 = 0.960, r pred 2 = 0.899) were used to establish the model, and the structure-activity relationship of the compounds was explained by the contour maps. The binding site was predicted by two different kinds of software, and the binding modes of the compounds were further explored. A series of key amino acids Val288, Ser289, Gly314, Thr624, Lys661 were found to play a key role in the activity of the compounds. Molecular dynamics (MD) simulations were carried out for compounds 04, 17, 21, and 35, which had different activities. The reasons for the activity differences were explained by the interaction between compounds and LSD1. The binding free energy was calculated by molecular mechanics generalized Born surface area (MM/GBSA). We hope that this research will provide valuable information for the design of new reversible LSD1 inhibitors in the future.

Expression, purification, and in vitro characterization of kinase domain of NtGCN2 from tobacco.

General control nonderepressible 2 (GCN2) can phosphorylate the α subunit of eukaryotic initiation factor eIF2 (eukaryotic translation initiation factor 2) to down-regulateprotein synthesis in response to various biotic and abiotic stresses. However, the kinase activity of plant GCN2 has not been well-characterized in vitro. In this study, the kinase domain of Nicotiana tabacum GCN2 (NtGCN2) was inserted into the pET15b vector for prokaryotic expressionin Escherichia coli BL21-CodonPlus-(DE3)-RIPL after induction by 0.5 mmol L-1 IPTG for 13 h at 16 °C. The soluble protein was collected and purified by Ni2+-NTA agarose column, anion exchange, and molecular sieve, and the purified proteinwas used for kinase assays and the preparation of a polyclonal antibody. Enzyme-linked immunosorbent assay results showed that the titer of the antiserum was 1:520K. Western blot analysis showed that the prepared antibody reacted with GCN2 in tobacco. Additionally, the kinase activity of NtGCN2 was characterized by using recombinant NteIF2α protein as a substrate in vitro. The results showed that NtGCN2 phosphorylated NteIF2α in vitro, with the level of phosphorylation positively correlated with the NtGCN2 concentration and reaction time. Our study has prepared a specific antibody, and proves NtGCN2 can phosphorylate NteIF2α in vitro, which lays a foundation for further study of the function and interaction network of NtGCN2.

Metagenomic insights into effects of wheat straw compost fertiliser application on microbial community composition and function in tobacco rhizosphere soil.

The application of fertilisers incorporated with plant residues improves nutrient availability in soils, which shifts the microbial community structure and favours plant growth. To understand the impact of wheat straw compost fertiliser on soil properties and microbial community structure, tobacco planting soils were treated with four different fertilisers using varied amounts of straw compost fertiliser and a no fertiliser control (CK). Results showed that different fertilisers affected available soil nutrient contents differently. Treatment of tobacco soil with application of combined chemical fertiliser/wheat straw compost led to improved soil chemical properties, and increased soil organic matter and available phosphorus and potassium content. Treatment with FT1 200 kg/mu straw was found to be superior in improving soil fertility. Metagenomic DNA sequencing revealed that different fertiliser treatments resulted in changes in the microbial community composition. In soil treated with FT2 300 kg/mu straw for 60 days, the predominant bacterial phyla were Proteobacteria, Actinobacteria, and Verrucomicrobia, whereas Cyanobacteria, Basidiomycota, and Chlorophyta were found in high abundance in soil samples treated with FT1 200 kg/mu straw for 30 days. Functional annotation of metagenomic sequences revealed that genes involved in metabolic pathways were among the most abundant type. PCoA analysis clearly separated the samples containing straw compost fertiliser and chemical fertiliser. A significant correlation between soil properties and the dominant phyla was identified.

Sarkosyl: A milder detergent than SDS for identifying proteins with moderately high hyperstability using gel electrophoresis.

Sodium dodecyl sulfate (SDS) is a detergent used as a strong denaturant of proteins in gel electrophoresis. It has previously been shown that certain hyperstable, also known as kinetically stable, proteins are resistant to SDS and thus require heating for their denaturation in the presence of SDS. Because of its high denaturing strength, relatively few proteins are resistant to SDS thereby limiting the current use of SDS-PAGE for identifying hyperstable degradation-resistant proteins. In this study, we show that sarkosyl, a milder detergent than SDS, is able to identify proteins with moderately high kinetic stability that lack SDS-resistance. Our assay involves running and subsequently comparing boiled and unheated protein samples containing sarkosyl, instead of SDS, on PAGE gels and identifying subsequent differences in protein migration. Our results also show that sarkosyl and SDS may be combined in PAGE experiments at varying relative percentages to obtain semi-quantitative information about a protein's kinetic stability in a range inaccessible by probing through native- or SDS-PAGE. Using protein extracts from various legumes as model systems, we detected proteins with a range of protein stability from nearly SDS-resistant to barely sarkosyl resistant.

Prevention and control of infectious diseases among primary schools in Xi’an / 中国学校卫生

Objective@#To understand prevention and control of infectious disease among primary schools in Xi’an. @*Methods@#A total of 93 primary schools were selected through multi-stage stratified sampling method from 14 counties in Xi’an, then these schools are surveyed to meaure the classroom ventilation.@*Results@#About 28.0% (26/93) of these schools met the national standard for health staffs, with 7.4%(4/54) in urban primary schools and 56.4% in rural primary schools (22/39), the difference between urban and rural areas is statistically significant(P<0.01). Urban primary schools were better than rural primary schools in morning inspection, registration and tracking for illness, verification of vaccination certificates, surveillance and reporting of epidemic outbreaks, verification of school recovery(P<0.05). The average per capita area among the 92 primary schools was (1.22±0.34) m2, with (1.08±0.26) m2 in urban primary schools and (1.42±0.33) m2 in rural primary schools, the difference between urban and rural areas was statistically significant (P<0.01). The average per capita ventilation area of 92 primary schools was (0.15±0.07) m2 with (0.13±0.04) m2 in urban primary schools and (0.19±0.08) m2 in rural primary schools, the difference between urban and rural areas was statistically significant (P<0.01).@*Conclusion@#School-based infectious diseases prevention and control needs to be improved in Xi’an, especially in health personnel. Urban schools appear superior in the prevention and control of infectious diseases and sanitary conditions, while rural schools appear superior in student density and classroom ventilation.

Overexpression of Tobacco GCN2 Stimulates Multiple Physiological Changes Associated With Stress Tolerance.

General control non-derepressible-2 (GCN2) is a ubiquitous protein kinase that phosphorylates the α subunit of the eukaryotic initiation factor, eIF2, preventing the initiation of a new cycle of protein synthesis, subsequently reducing the global protein biosynthesis. GCN2 can also regulate the response of plants to biotic and abiotic stresses. In this study, two GCN2 homologs, NtGCN2-1 and NtGCN2-2, were cloned from Nicotiana tabacum, and were predicted to have been derived from their progenitors in N. tomentosiformis and N. sylvestris, respectively. The phosphorylation of NteIF2α could be activated by promoting the expression of NtGCN2 with plant hormones, including salicylic acid (SA), azelaic acid (AZA), methyl jasmonate (MeJA), and by imposition of different stresses (Bemisia tabaci infection, drought, and cold), indicating that NtGCN2 is involved in the response of plants to multiple biotic and abiotic stresses. We also observed that the overexpression of NtGCN2-1 significantly influenced different physiological processes. It promoted seed germination and root elongation. The content of total soluble sugars and reducing sugars were decreased, whereas those of chlorophyll a and b were increased in the GCN2 overexpressing plants. In addition, the overexpressing plants had lower content of reactive oxygen species and exhibited higher antioxidant activities. These physiological alterations could be attributed to the changes in the endogenous phytohormones, decrease in the SA and abscisic acid content, and accumulation of MeJA and AZA. It indicated that the overexpression of NtGCN2 in tobacco, stimulated the plant defense responses via phosphorylation of NteIF2α and regulation of plant hormones, and changes in the antioxidant ability and plant nutrient status.

Fabrication of viable and functional pre-vascularized modular bone tissues by coculturing MSCs and HUVECs on microcarriers in spinner flasks.

Slow vascularization often impedes the viability and function of engineered bone replacements. Prevascularization is a promising way to solve this problem. In this study, a new process was developed by integrating microcarrier culture and coculture to fabricate pre-vascularized bone microtissues with mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs). Initially, coculture medium and cell ratio between MSCs and HUVECs were optimized in tissue culture plates concerning cell proliferation, osteogenesis and angiogenesis. Subsequently, cells were seeded onto CultiSpher S microcarriers in spinner flasks and subjected to a two-stage (proliferative-osteogenic) culture process for four weeks. Both cells proliferated and functioned well in chosen medium and a 1 : 1 ratio between MSCs and HUVECs was chosen for better angiogenesis. After four weeks of culture in spinner flasks, the microtissues were formed with high cellularity, evenly distributed cells and tube formation ability. While coculture with HUVECs exerted an inhibitory effect on osteogenic differentiation of MSCs, with downregulated alkaline phosphatase activity, mineralization and gene expression of COLI, RUNX2 and OCN, this could be attenuated by employing a delayed seeding strategy of HUVECs against MSCs during the microtissue fabrication process. CONCLUSION: Collectively, this work established an effective method to fabricate pre-vascularized bone microtissues, which would lay a solid foundation for subsequent development of vascularized tissue grafts for bone regeneration.

[A qualitative research on impact factors for the effectiveness of pit and fissure sealing in Xi'an city].

PURPOSE: To explore the factors influencing the effectiveness of pit and fissures sealing in order to provide suggestions to policy makers for quality promotion of pit and fissure program. METHODS: After developing outline of the qualitative interview, the method of focus group discussion was applied in this study. A total of 32 persons including dental clinical experts, dentists who operated pit and fissure sealing and program managers were interviewed. The data was collected, marked and classified by using MAXqda software. RESULTS: The main factors influencing the effectiveness of pit and fissure sealing in Xi'an city were the service providers, the service needers and the management of external organization. CONCLUSIONS: In order to promote the preventive effect of pit and fissure sealing, attention should be paid to increase investment from the government, promote school-based program mode, strengthen the supervision of designated medical institutions and the external management.

[Impact factors analysis on child neglect of children aged 3-6 year-old in rural areas of China].

OBJECTIVE: To investigate the major influence factors of child neglect among rural areas children aged 3-6 years in China. METHODS: According to multi-stage stratified cluster sampling, distribution characteristics and the level of economic development, we randomly sampled 10 provinces, 26 cities (8 capital cities, 16 prefecture-level cities and 2 municipalities) using lottery method. Depending on the different level of economic and cultural, we sampled one medium county from each city (municipalities sampled two counties), a total of 28 counties. All towns were divided into high, medium and low three levels in accordance with economic and cultural, each level sampled one town, each conty sampled three towns, a total of 84 towns were sampled. Each town sampled 40 children, including 20 boys and 20 girls (including each 10 children aged 3 to 6, half and half boys and girls). 4 096 rural children aged 3-6 years old were sampled and surveyed of China. Based on "the Neglect Norms for Children Aged 3 to 6 Years Old in Rural Areas of China" explored the risk factors of child neglect. SPSS 18. 0 for windows was employed for statistics analysis. Multifactorial analysis was conducted through multivariate Logistic Regression. RESULTS: The total neglected rate of the rural children aged 3 to 6 was 53.7% (2,047/3,810), and the total neglected degree was 44.42±7.57. The multivariate logistic regression analysis showed that, neglected risk of the boys was higher than the girls (OR=1.30, 95% CI: 1.13-1.49), neglected risk of children were higher, when their mothers were migrant workers (OR=2.18, 95% CI: 1.59-3.00); the lower educational level of mothers, the higher neglected risk of children, the children in families that mothers only received primary education or was uneducated were most likely to be neglected (OR=2.92, 95% CI: 1.91-4.47), the second was the families that mothers received primary education (OR=1.86, 95% CI: 1.29-2.69); the lower the household income, the easier to be neglected the children, the easiest one was the household income less than 5000 RMB (OR=2.85, 95% CI: 2.14-3.79), the second was 5000-9,999 RMB (OR=1.76, 95% CI: 1.40-2.20); the relationship between children and mothers was distanced, neglect risk of children was higher (OR=3.88, 95% CI: 1.31-11.52); left behind children were easier to be neglected (OR=1.30, 95% CI: 1.09-1.54). CONCLUSION: The influence factors among children aged 3-6 years in the rural areas of China were boys, mother's occupation, maternal education level, the relationship between children and mothers, household income and left-behind children.

Perfusion culture-induced template-assisted assembling of cell-laden microcarriers is a promising route for fabricating macrotissues.

Mass transfer limitation in conventional top-down tissue engineering makes it impossible to fabricate large size viable tissue replacements. In the present study, we aimed at performing a systemic investigation of the assembling process in perfusion culture for fabricating centimeter-scale macrotissues from cell-laden microcarriers following a bottom-up modular approach. Cells (human fibroblasts, human mesenchymal stem cells, or HepG2 cells) were seeded onto microcarriers (Cytopore-2 or CultiSpher S) in spinner flasks and cultured for 14 days and subsequently transferred to a perfusion chamber for assembling. It was found that growth of different cells on different microcarriers varied and aggregation of cell-laden microcarriers was favored with CultiSpher S. After perfusion culture for 14 days, while all microtissues could assemble into integral macrotissues, macrotissues of HepG2 cells were structurally most inferior and the assembling of cell-laden CultiSpher S led to a significant shrinkage. By designing perfusion chamber and using agar-based templates, tubular, disc, and alphabetic letter-shaped macrotissues could be easily fabricated, suggesting template-assisted assembling. Importantly, it was revealed that there existed both optimal perfusion culture time (21 days) and packing condition (1/4 compression) for the assembling of microtissues. This study lays a solid foundation for future applications of this microtissue assembling technique in tissue engineering.

[Development of national neglect norm for urban primary school students of China].

OBJECTIVE: To develop the national neglect norms for urban primary school students in China. METHODS: According to multi-stage stratified cluster sampling principle, 24 cities of 13 provinces (municipalities) in China were selected during December 1 to 31, 2008. A total of 1491 students in grade 1 - 3 and 2236 students in grade 4 - 6 were selected. Questionnaire was designed by authors and the final norms were determined through several statistical analysis methods, such as item analysis method, factor analysis method, reliability analysis method. The reliability analysis and validity analysis were used to test the stability and reliability of the norms. The evaluation criteria of the scale was determined by the percentile method, then the initial development of the norm was completed. RESULTS: The two questionnaires of grade 1 - 3 and grade 4 - 6 students consisted of 55 and 57 items, respectively, whose item loadings were ranged from 0.301 to 0.687 and 0.321 to 0.730, which met the statistical requirements. For grade 1 - 3 students, the scale's total Cronbach α coefficients was 0.914, the total split-half reliability coefficients was 0.896, the Cronbach α coefficients of four level was above 0.737 except medical and social neglect, split-half reliability was ranged from 0.461 to 0.757; for grade 4-6 students, the scale's total Cronbach α coefficients was 0.916, split-half reliability was 0.883, except social neglect, the Cronbach α coefficients of other level was ranged 0.457 to 0.856, split-half reliability was ranged from 0.500 to 0.798. The total neglect cut-off score of the two scales grade 1-3 and 4-6 were 125 and 155, respectively. CONCLUSION: The structure of two norms was reasonable. The scales have good stability and reliability.

[Development of the National Norms of Negligence on children aged 0 to 35 months in rural areas of China].

OBJECTIVE: To develop the National Norms of Negligence (NNN) for rural children aged 0 to 35 months. METHODS: According to multi-stage stratified cluster sampling principle, 10 provinces or municipalities (Jilin, Shanxi, Shanxi, Beijing, Anhui, Jiangsu, Hunan, Hubei, Yunnan, Chongqing) in China were selected. A national research group was formed collaboratively. A questionnaire was designed by ourselves. According to several statistical analysis methods, such as item, factor and reliability analysis etc. we determined the norm. The evaluation criteria of the scale were determined by percentile method. Finally, the reliability and validity of the norm were evaluated. RESULTS: In total, 2310 children were surveyed, in which the effective sample were 2227, with an effective rate as 96.4%. The scale consisted of 6 neglected dimensions and 65 items in total. The total Cronbach's a coefficient of the scale was 0.903, with the split-half reliability coefficient as 0.829, the parallel reliability as 0.720 and the re-test reliability as 0.678, respectively. The total neglect cut-off score of this scale was 139. CONCLUSION: The scale seemed to have perfect stability and reliability and all the statistical indicators met the psychometric demands.

[Comparative study on the situation of neglected children aged 3-6 year-olds between urban and rural areas of China].

OBJECTIVE: To investigate and analyze the situation of urban and rural neglected children aged 3 - 6, in China, so as to provide basis for the analysis and comparison on relevant risk factors. METHODS: 1163 urban children aged 3 - 6 (with 49.6% males and 4.5% with minority ethnicity) were investigated from 25 cities of 14 provinces, autonomous regions and municipalities in the whole country. Multi-stage stratified cluster sampling method was used. Again, using the same sampling method, 4096 rural children (of whom 50.6% were males with 6.2% as minorities) were chosen from 26 cities of 10 provinces or municipalities. Identification of children being neglected was based on "Child Neglect Evaluation Norms of Children Aged 3 - 6 Years in Urban/Rural China". SPSS-Windows 13.0 was employed for data analysis. Scores, frequency/degrees, age, sex and types (physical, emotional, educational, safety, medical and social) of children under negligence on every group of the regions, were calculated. χ(2) test (Chi-Square) and Analysis of variance (ANOVA) were processed to determine the significance of their differences. RESULTS: The overall frequencies of negligence were 28.0% and 53.7% respectively among the urban and rural children aged 3 - 6, while the total degrees of negligence were 42.2 and 44.4 respectively. Significant difference was found between children from the urban and the rural areas (P < 0.05). Significant difference was also found between urban and rural children on every age group (P < 0.05). The frequencies of negligence among males were 32.6% and 55.9% respectively in urban and rural areas while among females, the figures appeared to be 23.7% and 51.6% respectively. The degrees of negligence were 42.7 and 44.6 among male while 41.8 and 44.3 among female children, in the urban or rural areas. Significant differences were found on male or female between urban and rural groups (P < 0.05). Frequencies of negligence in urban children aged 3 - 6 for the six types were from 5.1% to 12.9%, with the frequency in rural areas as 13.1% - 26.6%. Significant difference was found between urban and rural group for any other type (P < 0.05), in addition to the safety type. The degrees of negligence in urban children aged 3 - 6 for the different type were between 39.4 and 43.4, while in the rural areas as from 36.5 to 48.2, with significant difference for every type (P < 0.05). The degrees of negligence related to education, emotion, or physical strength were more serious on children from the urban than from the rural areas. The highest frequency of child negligence was seen in the single-parent families on both urban and rural groups (42.9% and 60.0% respectively), with no significant difference found (P > 0.05). The urban and rural children aged 3 - 6 were mainly involved in single item of negligence, with incidence rates as 16.5% and 22.7% and proportions as 58.9% and 45.1% respectively, despite the factors as age or sex. CONCLUSION: There were large differences on the situation of negligence between the urban and rural children aged 3 - 6. The frequencies and degrees of negligence in every age group and different sex for children living in the rural areas were higher than those urban children. The frequency of negligence among boys was higher than girls for both urban and rural areas. The rural children had suffered more serious negligence than the urban children at any other type, in addition to the 'safety'. Both urban and rural children had the highest frequency of negligence in single-parent family, and were mainly suffered from single item of negligence.

[Development of national neglect norms for children aged 3 to 6 years in rural areas of China].

OBJECTIVE: To develop the national neglect norms for rural children aged 3 to 6 years, which are suitable for Chinese situations. METHODS: According to the multi-stage stratified cluster sampling principle, 84 towns of 10 provinces or municipalities were selected in China. Children aged 3 to 6 years were surveyed in November 2010, the sample of analysis were 3240 (of whom males were 49.6% (1608/3240) and the Han nationality were 93.3% (3023/3240)). Questionnaire was designed by authors and deleted items that did not meet the requirements through several statistical analysis methods, such as item analysis method, factor analysis method, reliability analysis method. The reliability analysis and validity analysis were used to test the stability and reliability of the norm. The evaluation criteria of the scale was determined by the percentile method, then the initial development of the norm completed. RESULTS: After deleting inappropriate items by statistical processing, finally, the scale consisted of 57 items, and included 6 neglected dimensions (physical neglect, emotional neglect, educational neglect, safe neglect, medical neglect and social neglect). Its item loadings ranged from 0.359 to 0.789, which met the statistical requirements. The scale's total Cronbach α coefficients 0.904, the total split-half reliability coefficients were 0.820, the 6 neglect dimensions' Cronbach α coefficients ranged from 0.620 to 0.815, the 6 neglect dimensions' split-half reliability coefficients ranged from -0.034 to 0.789, the scale's parallel reliability were 0.785 and it's re-test reliability were 0.613. After construct validity, external validity and content validity testing, the result showed that this scale could effectively reflect the real neglected status of children investigated. The total neglect cut-off score of this scale were 121. CONCLUSION: The scale has good stability and reliability. And it adapts Chinese conditions and it's convenient to operate.

[The status of child neglect among rural areas children aged 0 - 6 years in China].

OBJECTIVE: To explore the status of child neglect among rural areas children aged 0 - 6 years in China. METHODS: A total of 7411 rural children aged 0 - 6 years old who were composed of two age groups (3315 children aged 0 - 2 years old and 4096 children aged 3 - 6 years old) were multistage stratified randomly sampled from 84 villages which were representative of 10 provinces of China, in accordance with sex and age in November 2010. To identify the child neglect based on the Neglect Norms for Children Aged 0 to 2 and 3 to 6 Years Old in Rural Areas of China, SPSS 13.0 was employed for analyzing neglect frequency and degree for every group of different age, sex and neglect type (including physical, emotional, educational, medical, safety and social neglects). χ(2) test and analysis of variance were also used. RESULTS: The degree of child neglect for the children aged 0 - 2 years old was 45.01 ± 7.56, the neglect frequency was 54.9% (1819/3315); the degree of child neglect for the children aged 3 - 6 years old was 44.42 ± 7.57, the neglect frequency was 53.8% (2203/4096). The neglect frequency of children aged 0, 1, 2 years old were 58.5% (654/1117), 52.2% (597/1144), 53.9% (568/1054) (P < 0.05). For children aged 3 - 6 years old, the degrees of emotional and safety neglect for males (44.60 ± 7.86, 36.82 ± 9.03) were higher than females (44.03 ± 7.72, 36.25 ± 9.05) (P < 0.05); and the frequencies of emotional and social neglect for males (16.8% (349/2072), 28.3% (586/2072)) were also higher than females (14.1% (286/2024), 24.8% (503/2024)) (P < 0.05). All children of two age groups suffered neglect mainly on one of the six neglect types (incidences were 20.6% (683/3315) and 22.7% (931/4096)). For 0-2 age groups, the higher neglect frequencies happened in the single-parent family and the remarried family (62.5% (15/24) and 63.2% (12/19)), but for children aged 3 - 6 years old groups, it happened in the single-parent family (60.0%, 27/45). CONCLUSION: Degree and frequency of child neglect among children aged 0 to 6 years old in the rural areas of China are high, and we should find out risk factors and provide efficient prevention measures.

[The status of child neglect for children aged 3 - 17 years in China].

OBJECTIVE: To explore the status of child neglect among urban areas children and adolescents aged 3 - 17 years in China. METHODS: Using multi-stage stratified cluster sampling method, 8001 children and adolescents were recruited from 30 cities of 14 provinces in December 2008. The investigation was carried based on The 3 - 6 years old children Neglect Norms of city in China and The 6 - 17 years old children Neglect Norms of city in China. SPSS 13.0 was employed for analyzing neglect rate and degree for group of areas, age, sex and neglect type (including neglect of physical, emotional, educational, medical, safety and social). RESULTS: The degrees of child neglect for urban children aged 3 - 6 (children aged 6 were not in school), 6 - 8 (children aged 6 were school children), 9 - 11, 12 - 14 and 15 - 17 were 42.2 ± 6.9, 42.8 ± 8.4, 42.1 ± 9.1, 46.4 ± 9.2 and 49.7 ± 8.4(P < 0.05), the neglect rates were 28.0% (326/1163), 28.8% (431/1496), 27.2% (543/1962), 22.4% (373/1664) and 32.8% (563/1716) (P < 0.05), respectively. There were differences in six neglect types for each group; the neglect degrees and rates were high in the group aged 12 - 14 and 15 - 17 (46.4 ± 9.2 and 49.1 ± 8.4, 22.4% (373/1664) and 32.8% (563/1716), respectively); for each neglect type, the neglect degree of children aged 15 - 17 were highest (52.8 ± 8.0, 47.3 ± 11.6, 49.5 ± 10.8, 42.4 ± 10.3, 52.2 ± 16.0, 56.0 ± 10.1). The degree and rate of neglect in West were highest, Middle was next, the East was lowest (the neglect degrees were 45.4 ± 9.1, 45.0 ± 9.3 and 44.0 ± 8.8, P < 0.05;the rates were 30.7% (1340/4361), 25.0% (756/3024) and 21.2% (131/616), P < 0.05). Degree and rate of child neglect for children without siblings were lower than children with siblings (the neglect degrees were 44.2 ± 9.0 and 47.6 ± 9.1, P < 0.01; the rates were 26.1% (1572/6017) and 33.0% (655/1984), P < 0.01). Neglect degree of school children aged 6 years old (43.1 ± 7.3) was higher than children not in school (40.7 ± 7.8) (P < 0.01), but neglect rate was lower (the rates were 35.4% (60/169) and 21.3% (84/394), P < 0.01). CONCLUSION: Neglect degree and rate of urban children and adolescents aged 3 - 17 years in China were both high, and neglect deserved more attentions.

Quantifying the kinetic stability of hyperstable proteins via time-dependent SDS trapping.

Globular proteins are usually in equilibrium with unfolded conformations, whereas kinetically stable proteins (KSPs) are conformationally trapped by their high unfolding transition state energy. Kinetic stability (KS) could allow proteins to maintain their activity under harsh conditions, increase a protein's half-life, or protect against misfolding-aggregation. Here we show the development of a simple method for quantifying a protein's KS that involves incubating a protein in SDS at high temperature as a function of time, running the unheated samples on SDS-PAGE, and quantifying the bands to determine the time-dependent loss of a protein's SDS resistance. Six diverse proteins, including two monomer, two dimers, and two tetramers, were studied by this method, and the kinetics of the loss of SDS resistance correlated linearly with their unfolding rate determined by circular dichroism. These results imply that the mechanism by which SDS denatures proteins involves conformational trapping, with a trapping rate that is determined and limited by the rate of protein unfolding. We applied the SDS trapping of proteins (S-TraP) method to superoxide dismutase (SOD) and transthyretin (TTR), which are highly KSPs with native unfolding rates that are difficult to measure by conventional spectroscopic methods. A combination of S-TraP experiments between 75 and 90 °C combined with Eyring plot analysis yielded an unfolding half-life of 70 ± 37 and 18 ± 6 days at 37 °C for SOD and TTR, respectively. The S-TraP method shown here is extremely accessible, sample-efficient, cost-effective, compatible with impure or complex samples, and will be useful for exploring the biological and pathological roles of kinetic stability.