[Development of a risk assessment scale and test of its validity and reliability for venous thromboembolism in adult burn patients].

Objective: To develop a venous thromboembolism (VTE) risk assessment scale for adult burn patients and to test its reliability and validity. Methods: The scale research method and multi-center cross-sectional survey method were used. Based on the results of literature analysis method and brain-storming method, the letter questionnaire for experts was formulated. Then 27 experts (9 doctors of burn department, 9 vascular surgeons, and 9 nurses) were performed with two rounds of correspondences by Delphi method, and the reliability of the experts was analyzed. The weight of each item was determined by optimal sequence diagram method and expert importance evaluation to form the VTE Risk Assessment Scale for Adult Burn Patients. A total of 223 adult burn inpatients, who were admitted to 5 tier Ⅲ grade A general hospitals including the Affiliated Hospital of Southwest Medical University, West China Hospital of Sichuan University, the Affiliated Hospital of North Sichuan Medical College, Nanchong Central Hospital, and the Second People's Hospital of Yibin City from October 1st 2019 to January 1st 2020, were selected as respondents by convenience sampling method. The first assessment was performed with the VTE Risk Assessment Scale for Adult Burn Patients within 24 hours of admission of patients, and real-time assessment was performed as the patients' condition and treatment changed. The highest value was taken as the result. Correlation coefficient method and critical ratio method were used for item analysis; Cronbach's α coefficient was used to test the internal consistency of scale; content validity index was used to analyze the content validity of the scale, and receiver's operating characteristic (ROC) curve was drawn to test the predictive validity of the scale. Data were statistically analyzed with chi-square test, Pearson correlation analysis, independent sample t test, and Z test. Results: As four questionnaires in the first round of correspondence were rejected as unqualified, and another 4 experts were selected for the 2 rounds of correspondence. Most of them were aged 41 to 50 years with postgraduate degrees, engaging in the current profession for 11 to 30 years, and all of them had professional titles of associate senior or above. The scale, constructed through literature analysis, group brainstorming, and two rounds of correspondence, includes 3 primary items and 50 secondary items. In the first round of correspondence, the recovery rate of valid questionnaires and the ratio with expert opinions were 85.2% (23/27) and 47.8% (11/23), respectively. In the second round of correspondence, the recovery rate of valid questionnaires and the ratio with expert opinions were 100% (27/27) and 11.1% (3/27), respectively. The average collective authority coefficients of experts were both 0.90 in the 2 rounds of correspondence. The mean values of importance assignment, full score rate, and selection rate above 4 were 4.21, 52.5%, and 77.2%, respectively, in the first round of correspondence, and 4.28, 45.2%, and 85.8%, respectively, in the second round of correspondence. The mean coefficients of variation and the mean value of Kendall's coefficient of harmony for each item were 0.21 and 0.30 in the first round of correspondence, respectively, and 0.16 and 0.36 in the second round of correspondence, respectively. In the first and second rounds of correspondence, the Kendall's coefficients of harmony of 3 primary items (age and underlying diseases, burn injury factors, and burn treatment factors) and total secondary items were statistically significant (with χ2 values of 121.46, 107.09, 116.00, 331.97, 169.97, 152.12, 141.54, and 471.70, P<0.01). The weights of primary items for age and underlying diseases, burn injury factors, and burn treatment factors were 0.04, 0.05, and 0.07, respectively. The weights of secondary items ranged from 0.71 to 0.99, with assigned values of 3 to 6. The total burn area of 223 patients ranged from 1% to 89% total body surface area, and the patients were aged from 19 to 96 years, with the risk assessment score from 0 to 98. Nine patients developed VTE, with a risk assessment score of 41 to 90. The scores of 37 items were significantly positively correlated with the total score of scale (with r values of 0.14 to 0.61, P<0.05 or P<0.01), and the items were retained. There were 36 secondary items with statistically significant differences between the patients in high-score group and low-score group (with Z values of -4.88 to -2.09, t values of -11.63 to -2.09, P<0.05 or P<0.01), and the items were retained. The total Cronbach's α coefficient of scale was 0.88. The total content validity index of scale was 0.95. The optimal threshold of the scale for the diagnosis of VTE was 40, at which the sensitivity was 88.9%, the specificity was 87.4%, the Youden index was 0.87, and the area under the ROC curve was 0.96 (with 95% confidence interval of 0.93 to 0.99, P<0.01). Conclusions: The age and underlying diseases, burn injury factors, and burn treatment factors are the risk factors for VTE in adult burn patients. The VTE risk assessment scale for adult burn patients developed based on these factors has good reliability and validity, and provide good reference value for clinical VTE risk assessment.

["Xingnao Kaiqiao" Needling Stimulation Promotes Resuscitation of General Anesthesia Patients Undergoing Anodynia Gastrointestinal Endoscopy].

OBJECTIVE: To observe the effect of "Xingnao Kaiqiao" needling on the recovery of general anesthesia in mid-aged and elderly patients undergoing painless gastrointestinal endoscopy. METHODS: A total of 140 patients were randomly divided into control and treatment groups, with 70 cases in each group. After termination of the endoscopy, patients of the treatment group received acupuncture stimulation of Neiguan (PC 6), Shuigou (GV 26) and Sanyinjiao (SP 6) with sterilized disposable filiform needles which were manipulated with twirling-reducing method for 3 min in PC 6, with lifting-thrusting reinforcing method in SP 6, and with bird-peck needling in GV 26. The patients of the control group were not given any treatment. Before, the immediate time of termination of the endoscope examination, resuscitation and 10 min after the endoscope examination, the mini-mental state examination (MMSE) scores were obtained to assess the patients' recognition function. The Bi-spectral Index (BIS) values were recorded to assess the patients' depth of sedation and anesthesia, and the Observer's Assessment of Alertness/Sedation Scale (OAA/S) was used to evaluate the resuscitation/sedation level. RESULTS: Compared with the control group, the MMSE score of the treatment group was significantly higher at the 10th min after endoscope examination (P<0.05),suggesting an improvement of the cognitive function after acupuncture intervention. The duration from general anesthetic state (BIS values=40-60 points) to mild sedation (BIS values=80-90 points) was significantly shorter in the treatment group than in the control group (P<0.05). No significant differences were found between the two groups in the OAA/S score at the 10th min after endoscope (P>0.05). CONCLUSIONS: "Xingnao Kaiqiao" needling stimulation has a significant effect on the recovery of middle-aged and elderly patients undergoing painless gastroscopy during general anesthesia.

No causal effect of serum urate on bone-related outcomes among a population of postmenopausal women and elderly men of Chinese Han ethnicity--a Mendelian randomization study.

SUMMARY: We conducted a Mendelian randomization analysis to assess the effect of serum uric acid on bone-related outcomes using a weighted urate transporter genetic risk score as the instrumental variable. The results showed no significance. Our study identified no evidence of a causal role between uric acid and bone-related outcomes. INTRODUCTION: Observational studies have associated elevated levels of serum uric acid (SUA) with increasing bone mineral density (BMD) and a lowered prevalence of osteoporotic fractures (OFs) in postmenopausal women and elderly men. However, due to unmeasured confounding variables, these observational studies have not provided insight into the causal relationship between SUA and bone-related outcomes. Our aim was to evaluate the effect of SUA on bone-related outcomes using Mendelian randomization. METHODS: We recruited 1322 Chinese Han individuals (214 elderly men and 1108 postmenopausal women) from the Shanghai area in China. Mendelian randomization using a two-stage least-squares regression method was conducted with SUA as the exposure variable, a weighted urate transporter genetic risk score as the instrumental variable, and all-site BMD, bone turnover markers, and levels of 25-hydroxyvitamin D3 [25(OH)D], serum calcium (Ca), serum phosphorus (P), and parathyroid hormone (PTH) as outcome variables. RESULTS: Strong associations between SUA and bone-related outcomes were observed in an ordinary observational analysis (lumbar spine: beta = 0.122, p < 0.0001; hip: beta = 0.104, p < 0.0001; femoral neck: beta = 0.108, p < 0.0001). However, the Mendelian randomization analysis showed no evidence for a causal association of SUA with BMD (lumbar spine: beta = 0.385, p = 0.257; hip: beta = 0.191, p = 0.499; femoral neck: beta = 0.194, p = 0.533). Similar results were found between SUA and other bone-related phenotypes. CONCLUSIONS: Our study identified no evidence of a causal role between SUA and bone-related outcomes, although strong associations in an observational analysis were observed in a population of postmenopausal women and elderly men.

Genomic identification of group A bZIP transcription factors and their responses to abiotic stress in carrot.

The basic-region/leucine-zipper (bZIP) family is one of the major transcription factor (TF) families associated with responses to abiotic stresses. Many members of group A in this family have been extensively examined and are reported to perform significant functions in ABA signaling as well as in responses to abiotic stresses. In this study, 10 bZIP factors in carrot were classified into group A based on their DNA-binding domains. The cis-acting regulatory elements and folding states of these 10 factors were analyzed. Evolutionary analysis of the group A members suggested their importance during the course of evolution in plants. In addition, cis-acting elements and the folding state of proteins were important for DNA binding and could affect gene expression. Quantitative RT-PCR was conducted to investigate the stress response of 10 genes encoding the group A factors. Six genes showed responses to abiotic stresses, while four genes showed other special phenomenon. The current analysis on group A bZIP family TFs in carrot is the first to investigate the TFs of Apiaceae via genome analysis. These results provide new information for future studies on carrot.

Structural and physiological characterization during wheat pericarp development.

KEY MESSAGE: The cytological and physiological features of developing wheat pericarp were clearly characterized in this report. Our results may be helpful to articulate the functions of pericarp during the seed development. Although wheat pericarp has been well studied, knowledge of the sequence of events in the process of pericarp development is incomplete. In the present study, the structural development process of wheat (Triticum aestivum L.) pericarp was investigated in detail using resin microtomy and microscopy. Chlorophyll contents, and photosynthetic and respiratory rates, in pericarp were determined using spectrophotometer and an oxygen electrode, respectively. Mineral nutrient contents were also determined using scanning electron microscopy. The main results are as follows: (1) based on the structures and physiological characteristics observed, the developmental process of pericarp was divided into four stages, growth, formation, extinction and maturation stages, pericarp exhibited specific features at each stage. (2) Pericarp development differed in different parts, or varieties, of wheat. The dorsal pericarp had fewer starch grains and slower rates of apoptosis than the abdominal mesocarp. The cross cells in dorsal pericarp had an irregular outline. When compared with soft wheat cv. Yangmai 11, mesocarp cells in hard wheat cv. Xumai 30 had more starch grains, larger cell size and longer development duration. (3) The chlorophyll content, photosynthesis rate and respiratory rate in pericarp increased gradually, reaching a maximum about 16 days after anthesis, and later decreased continually. The photosynthetic rate in pericarp was lower than the respiration rate. (4) The contents of mineral elements in pericarp, such as calcium, zinc, iron and potassium were higher than those in the inner endosperm. The data indicate that wheat pericarp has many functions, e.g. protection, photosynthesis, mineral accumulation, synthesis and degradation of starch.

High level expression of a recombinant acid phytase gene in Pichia pastoris.

AIMS: To achieve high phytase yield with improved enzymatic activity in Pichia pastoris. METHODS AND RESULTS: The 1347-bp phytase gene of Aspergillus niger SK-57 was synthesized using a successive polymerase chain reaction and was altered by deleting intronic sequences, optimizing codon usage and replacing its original signal sequence with a synthetic signal peptide (designated MF4I) that is a codon-modified Saccharomyces cerevisiae mating factor alpha-prepro-leader sequence. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene promoter with the MF4I- or wild type alpha-signal sequence were used to transform Pichia pastoris. The P. pastoris strain that expressed the modified phytase gene (phyA-sh) with MF4I sequence produced 6.1 g purified phytase per litre of culture fluid, with the phytase activity of 865 U ml(-1). The expressed phytase varied in size (64, 67, 87, 110 and 120 kDa), but could be deglycosylated to produce a homogeneous 64 kDa protein. The recombinant phytase had two pH optima (pH 2.5 and pH 5.5) and an optimum temperature of 60 degrees C. CONCLUSIONS: The P. pastoris strain with the genetically engineered phytase gene produced 6.1 g l(-1) of phytase or 865 U ml(-1) phytase activity, a 14.5-fold increase compared with the P. pastoris strain with the wild type phytase gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The P. pastoris strain expressing the modified phytase gene with the MF4I signal peptide showed great potential as a commercial phytase production system.

A delta-endotoxin encoded in Pseudomonas fluorescens displays a high degree of insecticidal activity.

The short field-life of Bacillus thuringiensis (Bt) insecticidal crystal protein has limited its use. When the Bt toxin is produced in Pseudomonas fluorescens it can be encapsulated and retain its effectiveness for two to three times longer than other Bt formulations. In order to improve Bt expression, we have synthesized cryIA(c) Bt delta-endotoxin encoding region (GenBank AF537267) according to the usage codon of P. fluorescens and transformed the Bt toxin expression cassette into P. fluorescens strains. T7 RNA polymerase and the T7 promoter system were used to control expression of Bt toxin. SDS-PAGE and Western blotting assay revealed that the delta-endotoxin was expressed as 8% of the total protein in P. fluorescens. In in vitro tests, release of toxin from dead bacteria was demonstrated. Supplementation of diets with Bt toxin-containing Pseudomonas bacterium resulted in high mortality of cabbage butterfly ( Pieris brassicae) larvae.

Molecular characterization of the iron-hydroxamate uptake system in Staphylococcus aureus.

To investigate iron uptake, a chromosomal locus containing three consecutive open reading frames, designated fhuC, fhuB, and fhuD, was identified in Staphylococcus aureus. Whereas the fhuC gene encodes an ATP-binding protein, fhuB and fhuD code for ferrichrome permeases and thus resemble an ATP-binding cassette transporter. A fhuB knockout mutant showed impaired uptake of iron bound to the siderophores but not of ferric chloride, suggesting that this operon is specific for siderophore-mediated iron uptake.

[Construction and expression of a new humanized salmon calcitonin variant].

OBJECTIVE: To study the reduction of immunogenicity of salmon calcitonin (sCT). METHODS: A new (11-17) humanized salmon calcitonin variant gene (11-17) hsCT was designed and synthesized to construct recombinant expression vector and express in E.coli G1724 with tryptophan. The fusion protein of humanized salmon calcitonin and thioredoxin (Trx-hsCT) obtained from the bacterial by means of osmotic pressure; The bioactivity of Trx-hsCT was estimated by serum calcium reduction method in rat; The quantitative Western blot analysis was used for detecting the immunogenicity of hsCT. RESULTS: Recombinant expressing vector pTrxFus-hsCT was expressed in high level and soluble in E.coli G1724, reaching 46% of total bacterial protein and obtaining the fusion protein of 90% purity; Special bioactivity of hsCT was preserved that of half pre-sCT and its immunogenicity reduced over 50% that of pre-sCT. CONCLUSION: Obtain a new humanized salmon calcitonin variant(11-17) hsCT which maintains a rather high bioactivity over pre-sCT and decreases distinctly its immunogenicity.

Effect of heparin on apoptosis in human nasopharyngeal carcinoma CNE2 cells.

In order to study the mechanism of the effect of heparin on apoptosis in carcinoma cells, the nasopharyngeal carcinoma cell line CNE2 was used to identify the effect of heparin on apoptosis associated with the expression of c-myc, bax, bcl-2 proteins by use of Hoechst 33258 staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), agarose gel electrophoresis, and flow cytometry, as well as Western blot analysis. The results showed that heparin induced apoptosis of CNE2 cells including the morphologic changes such as reduction in the volume, and the nuclear chromatin condensation, as well as the "ladder pattern" revealed by agarose gel electrophoresis of DNA in a concentration-dependent manner. The number of TUNEL-positive cells was dramatically increased to 33.6+/-1.2% from 2.8+/-0.3% by treatment with heparin in different concentrations (10 to approximately 40 kU/L). The apoptotic index was increased to 32.5% from 3.5% by detecting SubG1 peaks on flow cytometry. Western blot analysis showed that levels of bcl-2, bax and c-myc were significantly overexpressed by treatment with the increase of heparin concentrations. These results suggest that heparin induces apoptosis of CNE2 cells, which may be regulated by differential expression of apoptosis-related genes.

The EmrR protein represses the Escherichia coli emrRAB multidrug resistance operon by directly binding to its promoter region.

EmrR negatively regulates the transcription of the multidrug resistance pump-encoding operon, emrRAB, by binding to its regulatory region. The binding site spans the promoter and the downstream sequence up to the transcriptional start site of the operon. Structurally unrelated drugs that induce the pump interfere with this binding.

ZntR is an autoregulatory protein and negatively regulates the chromosomal zinc resistance operon znt of Staphylococcus aureus.

A chromosomally encoded znt operon of Staphylococcus aureus consists of two consecutive putative genes designated zntR and zntA. The zntA gene encodes a transmembrane protein that facilitates extrusion of Zn2+ and Co2+, whereas the zntR gene encodes a putative regulatory protein that controls the expression of the znt operon. The zntR gene was amplified using the polymerase chain reaction, cloned into Escherichia coli for overexpression as His-tagged ZntR and purified by Ni2+-affinity column. His-tag-free ZntR was purified to near homogeneity after digestion with enterokinase. Electrophoretic mobility shift assays (EMSAs) indicated that the ZntR bound to a fragment of DNA corresponding to the chromosomal znt promoter region with an affinity of about 8.0 x 10-12 M. The addition of 25 microM Zn2+ or Co2+ in the binding reaction completely or significantly inhibited association of ZntR with the znt promoter. DNase I footprinting assays identified a ZntR binding site encompassing 49 nucleotides in the znt promoter region that contained repeated TGAA sequences. These sequences have been proposed to be the binding sites for SmtB, a metallorepressor protein from the cyanobacterium Synechococcus, to its corresponding operator/promoter. In vitro transcription assays, using S. aureus RNA polymerase, revealed that ZntR represses transcription from the znt promoter in a concentration-dependent fashion. The EMSAs, DNase I footprinting and in vitro transcription assays indicate that ZntR is a trans-acting repressor protein that binds to the znt promoter region and regulates its own transcription together with that of zntA.

Molecular characterization of a chromosomal determinant conferring resistance to zinc and cobalt ions in Staphylococcus aureus.

A DNA fragment conferring resistance to zinc and cobalt ions was isolated from a genomic DNA library of Staphylococcus aureus RN450. The DNA sequence analysis revealed two consecutive open reading frames, designated zntR and zntA. The predicted ZntR and ZntA showed significant homology to members of ArsR and cation diffusion families, respectively. A mutant strain containing the null allele of zntA was more sensitive to zinc and cobalt ions than was the parent strain. The metal-sensitive phenotype of the mutant was complemented by a 2.9-kb DNA fragment containing zntR and zntA. An S. aureus strain harboring multiple copies of zntR and zntA showed an increased resistance to zinc. The resistance to zinc in the wild-type strain was inducible. Transcriptional analysis indicated that zntR and zntA genes were cotranscribed. The zinc uptake studies suggested that the zntA product was involved in the export of zinc ions out of cells.

Cloning and nucleotide sequencing of a Staphylococcus aureus gene encoding a branched-chain-amino-acid transporter.

We recently characterized a transposon-induced NaCl-sensitive mutant of Staphylococcus aureus (U. Vijaranakul, M. J. Nadakavukaren, D. O. Bayles, B. J. Wilkinson, and R. K. Jayaswal, Appl. Environ. Microbiol. 63:1889-1897, 1997). To further characterize this mutant, we determined the nucleotide sequence at the insertion site of the transposon on the S. aureus chromosome. Nucleotide sequencing revealed a 1,326-bp open reading frame (ORF442) encoding a hydrophobic 442-amino-acid polypeptide with a calculated molecular mass of 49,058 Da. The hydrophilicity profile of the gene product revealed the existence of 12 hydrophobic domains predicted to form membrane-associated alpha-helices. Comparison of the amino acid sequence of ORF442 with amino acid sequences in the GenBank database showed extensive homology with the branched-chain-amino-acid transport genes of gram-positive and gram-negative bacteria. This is the first brnQ gene in staphylococci to be described.