[Developmental effects of TCIPP and TnBP on zebrafish (Danio rerio) embryos].

Objective: To investigate the toxicity of tris (2-chloropropyl) phosphate (TCIPP) and tributyl phosphate (TnBP) on the growth and development of zebrafish embryos, as well as to explore the underlying mechanisms at the transcriptional level. Methods: With zebrafish as a model, two hpf zebrafish embryos were exposed to TCIPP and TnBP (0.1, 1, 10, 100, 500, and 1 000 µmol/L) using the semi-static method, and their rates of lethality and hatchability were determined. The transcriptome changes of 120 hpf juvenile zebrafish exposed to environmentally relevant concentrations of 0.1 and 1 µmol/L were measured. Results: The 50% lethal concentrations (LC50) of TCIPP and TnBP for zebrafish embryos were 155.30 and 27.62 µmol/L (96 hpf), 156.5 and 26.05 µmol/L (120 hpf), respectively. The 72 hpf hatching rates of TCIPP (100 µmol/L) and TnBP (10 µmol/L) were (23.33±7.72)% and (91.67±2.97)%, which were significantly decreased compared with the control group (P<0.05). Transcriptome analysis showed that TnBP had more differential genes (DEGs) than TCIPP, with a dose-response relationship. These DEGs were enriched in 32 pathways in total, including those involved in oxidative stress, energy metabolism, lipid metabolism, and nuclear receptor-related pathways, using the IPA pathway analysis. Among them, three enriched pathways overlapped between TCIPP and TnBP, including TR/RXR activation and CAR/RXR activation. Additionally, DEGs were also mapped onto pathways of LXR/RXR activation and oxidative stress for TnBP exposure only. Conclusion: Both TCIPP and TnBP have growth and developmental toxicities in zebrafish embryos, with distinct biomolecular mechanisms, and TnBP has a stronger effect than TCIPP.

[Measuring laryngotracheal stenosis by extracting centerline based on CT 3D reconstruction].

Objective: To compare the accuracy of the centerline extracted based on CT 3D reconstruction and conventional CT 3D reconstruction in measuring the length and degree of laryngotracheal stenosis. Methods: A retrospective analysis was performed on 35 patients with laryngotracheal stenosis (including 19 cases without tracheotomy and 16 cases with tracheotomy) treated in the Department of Otorhinolaryngology Head and Neck Surgery of the First Affiliated Hospital of Guangxi Medical University from March 2006 to March 2016, including 20 males and 15 females, whose ages ranged from 1 to 73 years, with a median age of 40.5 years. And CT data of 20 normal subjects were included in the same period, including 10 males and 10 females, whose ages ranged from 20 to 63 years, with a median age of 37.0 years. The continuous cross-sectional area of the airway perpendicular to the centerline was obtained by Mimics software. The area was compared with the discontinuous cross-sectional areas reconstructed by conventional CT 3D reconstruction software advantage workstation, also the length of cervical trachea, the length of stenosis, and the minimum airway area were compared. Multi-factor linear stepwise regression method was used to analyze the factors influencing the measuring difference between the two methods. Three patients with laryngotracheal stenosis were selected, and the measured stenosis length was compared with the surgical specimens to evaluate the accuracy of the two methods. SPSS 26.0 software was used for statistical analysis. Results: In normal people, the areas of thyroid cartilage notch, glottis, inferio thyroid cartilage margin, inferio cricoid cartilage margin, and suprasternal notch planes measured by Mimics centerline method were smaller than those measured by conventional CT 3D reconstruction (t thyroid cartilage notch=4.685, tglottis=3.791, tlower thyroid cartilage margin=5.621, tlower cricoid cartilage margin=6.312, tsuprasternal notch plane=6.436, P<0.05). And the airway length measured by Mimics centerline method from the inferior thyroid cartilage to the superior sternal notch was longer (t=9.79, P<0.001). In laryngotracheal stenosis, in the non-tracheotomy group, the minimum airway area measured by Mimics centerline method was smaller and the stenosis length was longer than those measured by the conventional CT 3D reconstruction, and the difference was statistically significant (tminimum airway area=2.562, tstenosis length=5.240, P<0.05). In the tracheotomy group, the stenosis length measured by Mimics centerline method was longer than that measured by conventional CT 3D reconstruction, and the difference was statistically significant (tstenosis length=2.854, P<0.05). Multi-factor linear regression analysis showed that different CT thickness had a statistically significant effect on the difference in the length of stenosis measured by the two methods (b=-5.370, t=-3.306, P=0.004), and different tracheal forward angle had a statistically significant effect on the difference in the minimum airway area measured by the two methods (b=-0.419, t=-2.208, P=0.04). The difference between the measured length of the Mimics centerline method and the intraoperative specimens was less than 0.5 mm. Conclusion: The centerline extracted based on CT 3D reconstruction can precisely reflect the laryngotracheal morphology and measure laryngotracheal stenosis more accurately.

[Application of the five-level pediatric emergency triage system: a single center study].

Objective: To assess the effectiveness in optimizing resources and shortening critical children's waiting time in pediatric emergency department (PED) with five-level pediatric emergency triage system (PETS). Methods: This retrospective study was conducted in the First Affiliated Hospital of Xiamen University after PETS was applied. The data of patients who visited the pediatric emergency department from January 2015 to December 2017 were collected and analyzed, including age, sex, diseases, visiting time, triage rate and destination. Results: A total of 375 985 patients were included, among whom males were 225 308 (59.9%) and females were 150 677 (40.1%), all younger than 14 years of age. The number of critical cases (level Ⅰ, level Ⅱ and level Ⅲ) was increased from 4 719 (3.7%) in 2015, 12 209 (10.2%) in 2016 to 16 188 (12.7%) in 2017. The number of non-critical patients (level Ⅴ) decreased year by year, as from 98 213 (76.8%) in 2015 to 75 210 (62.6%) in 2016 and 78 857 (61.7%) in 2017. The patients who classified as level Ⅰ or levelⅡaccording to the PETS were seen immediately by physician (n=1855, 0.5%). Overall, 119 738 patients (98.3%) who were classified as level Ⅲ or level Ⅳ could be seen by physician in a timely manner according to triage guidelines, while 2 112 patients (1.7%) could not. The mean waiting time was 9.09 min in level Ⅲ, 17.7 min in level Ⅳ, and 55.76 min in level Ⅴ patients, respectively. The critical cases admitted to the intensive care units were 175 (36.2%) in 2015, 350 (62.8%) in 2016 and 374 (66.2%) in 2017. The etiologies were respiratory diseases (73.3%), gastrointestinal diseases (15.8%) and infectious diseases (3.1%). Conclusion: The application of PETS could optimize emergency resources and shorten the waiting time of critically ill children.

[Application of the computer-based respiratory sound analysis system based on Mel-frequency cepstral coefficient and dynamic time warping in healthy children].

OBJECTIVE: We designed a computer-based respiratory sound analysis system to identify pediatric normal lung sound. To verify the validity of the computer-based respiratory sound analysis system. METHOD: First we downloaded the standard lung sounds from the network database (website: http: //www.easyauscultation.com/lung-sounds-reference-guide) and recorded 3 samples of abnormal loud sound (rhonchi, wheeze and crackles) from three patients of The Department of Pediatrics, the First Affiliated Hospital of Xiamen University. We regarded such lung sounds as"reference lung sounds". The"test lung sounds"were recorded from 29 children form Kindergarten of Xiamen University. we recorded lung sound by portable electronic stethoscope and valid lung sounds were selected by manual identification. We introduced Mel-frequency cepstral coefficient (MFCC) to extract lung sound features and dynamic time warping (DTW) for signal classification. RESULT: We had 39 standard lung sounds, recorded 58 test lung sounds. This computer-based respiratory sound analysis system was carried out in 58 lung sound recognition, correct identification of 52 times, error identification 6 times. Accuracy was 89.7%. CONCLUSION: Based on MFCC and DTW, our computer-based respiratory sound analysis system can effectively identify healthy lung sounds of children (accuracy can reach 89.7%), fully embodies the reliability of the lung sounds analysis system.

Overexpressed miR-155 is associated with initial presentation and poor outcome in Chinese pediatric acute myeloid leukemia.

OBJECTIVE: Acute myeloid leukemia (AML) is the second leading leukemia in children. There is growing evidence that microRNAs (miRNAs) are crucial regulators involved in leukemogenesis. This study aimed to investigate the role of miR-155 in Chinese pediatric AML by evaluating its diagnostic and prognostic significance. PATIENTS AND METHODS: The expression of miR-155 and miR-25 in bone marrow specimens from 83 AML and 29 non-malignancies children were analyzed by TaqMan probe-based real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). RESULTS: The expression level of miR-155 was significantly higher in AML patients than in controls. Besides, a lowest miR-155 level was found in favorable prognosis group and t (15; 17)/M3 subgroup compared to the rest, while a higher level in C-Kit/FLT3-ITD mutation and relatively lower level existed in "Negative" mutation group. Moreover, miR-155 level was positively associated with the white blood cell (WBC) count, serum lactate dehydrogenase (LDH) and C-reaction protein (CRP) value in peripheral blood (PB), as well as miR-25/miR-196b expression levels. Survival analysis showed a statistically negative association with overall survival (OS) in the expression of miR-155 in chemotherapy group. CONCLUSIONS: These finding suggested that miR-155 expression cannot only be promising biomarker for the early detection of pediatric AML but also predict poor outcome. MiR-155 would be a novel biomarker for diagnosis, prognosis and therapy in pediatric AML.

Global convergence of Oja's subspace algorithm for principal component extraction.

Oja's principal subspace algorithm is a well-known and powerful technique for learning and tracking principal information in time series. A thorough investigation of the convergence property of Oja's algorithm is undertaken in this paper. The asymptotic convergence rates of the algorithm is discovered. The dependence of the algorithm on its initial weight matrix and the singularity of the data covariance matrix is comprehensively addressed.

Global analysis of Oja's flow for neural networks.

A detailed study of Oja's learning equation in neural networks is undertaken in this paper. Not only are such fundamental issues as existence, uniqueness, and representation of solutions completely resolved, but also the convergence issue is resolved. It is shown that the solution of Oja's equation is exponentially convergent to an equilibrium from any initial value. Moreover, the necessary and sufficient conditions are given on the initial value for the solution to converge to a dominant eigenspace of the associated autocorrelation matrix. As a by-product, this result confirms one of Oja's conjectures that the solution converges to the principal eigenspace from almost all initial values. Some other characteristics of the limiting solution are also revealed. These facilitate the determination of the limiting solution in advance using only the initial information. Two examples are analyzed demonstrating the explicit dependence of the limiting solution on the initial value. In another respect, it is found that Oja's equation is the gradient flow of generalized Rayleigh quotients on a Stiefel manifold.

The energy expenditure and nutritional status of college students. I. The energy cost and the total energy expenditure per day.

The energy cost of major activities was determined in healthy students. Among the 606 medical students, 319 were males and 287 were females. Their ages ranged from 18 to 24 years. Douglas' method was used to measure energy cost of each of a total of 42 activities, as well as that of the basal metabolic rates (BMR), resting metabolic rates (RMR) and the total energy expenditure per day under normal situations. The average RMR of male and female subjects were 0.669 +/- 0.033 and 0.656 +/- 0.030 kcal/sq.m/min respectively. The total energy expenditure per day of male students were 2706 kcal, and 2373 kcal for female students. The energy cost of single activities can be used as the basal data in studies of energy metabolism.

Study on iron nutritional status in adolescence.

A study on iron-deficiency anemia (IDA) in adolescence was conducted among 478 teen-age students in Shanghai. The study indicated that the intake of nutrients among the students was generally insufficient. The lack of protein, calcium, Vitamin A, Vitamin B1, and Vitamin B2 was more serious. The morbidities of IDA among male and female students were 15.8 and 32.6%, respectively, higher in the female group (P less than 0.01). The iron-deficiency sufferers among male and female students were 46.8 and 61.8%, respectively, also higher in the female group (P less than 0.01). The causes of IDA were analyzed by the method of stepwise regression. In a study of the effect of IDA on intelligence and physical development in adolescents, we found that there was no significant effect of IDA on intelligence quotient (IQ) and school performance. However, the speed and endurance capabilities of students of both sexes were correlated directly with hemoglobin level. In female students, the speed capability was correlated directly with the serum ferritin content. On the basis of these findings, a special 3-month school lunch program was initiated. The results indicate that a comprehensive, rational, and balanced diet is beneficial to hemoglobin, free erythrocyte porphyrin, and serum ferritin contents and improves adolescent development.

A recombinant fusion protein and DNA vaccines against foot-and-mouth disease virus type Asia 1 infection in guinea pigs.

On the basis of amino acid (aa) sequence of the tandem repeat 133-158-20-34-133-158 which consisted of aa 133-158 of VP1 and aa 20-34 of VP4 of Foot-and-mouth disease virus (FMDV) type Asia 1 a recombinant prokaryotic expression vector pAS1-P encoding a fusion protein and eukaryotic expression vectors pAS1-E and pAS1-EdeltaCpG-ODN representing DNA vaccines were constructed. Guinea pigs immunized with these vaccines showed both neutralizing antibody and T cell proliferation responses. FMDV challenge tests for the first time showed that the recombinant fusion protein and pAS1-E and pAS1-EdeltaCpG-ODN vaccines protected 86%, 60% and 43% of guinea pigs from FMDV type Asia1 challenge, respectively. The results also indicated that the immune response of animals treated with the vector pAS1-E containing an oligodeoxynucleotide (ODN), which consisted of immunostimulatory cytosine-phosphate-guanosine (CpG) motifs, was augmented by CpG ODN.

Immunogenicity of a recombinant fusion protein of tandem repeat epitopes of foot-and-mouth disease virus type Asia 1 for guinea pigs.

In this study, the sequences of capsid protein VPI regions of YNAs1.1 and YNAs1.2 isolates of foot-and-mouth disease virus (FMDV) were analyzed and a peptide containing amino acids (aa) 133-158 of VP1 and aa 20-34 of VP4 of FMDV type Asia I was assumed to contain B and T cell epitopes, because it is hypervariable and includes a cell attachment site RGD located in the G-H loop. The DNA fragments encoding aa 133-158 of VP1 and aa 20-34 of VP4 of FMDV type Asia 1 were chemically synthesized and ligated into a tandem repeat of aa 133-158-20 approximately 34-133-158. In order to enhance its immunogenicity, the tandem repeat was inserted downstream of the beta-galactosidase gene in the expression vector pWR590. This insertion yielded a recombinant expression vector pAS1 encoding the fusion protein. The latter reacted with sera from FMDV type Asia 1-infected animals in vitro and elicited high levels of neutralizing antibodies in guinea pigs. The T cell proliferation in immunized animals increased following stimulation with the fusion protein. It is reported for the first time that a recombinant fusion protein vaccine was produced using B and T cell epitopes of FMDV type Asia 1 and that this fusion protein was immunogenic. The fusion protein reported here can serve as a candidate of fusion epitopes for design of a vaccine against FMDV type Asia 1.

[Changes of the release of atrial natriuretic factor (ANF) and its gene transcription in morphine tolerance rat atrium].

Specific radioimmunoassay and molecular hybridization technique were used in the present work. Compared with the control rats, we found for the first time a significant decrease of ANF in plasma and a marked increase of ANF in atria in morphine tolerant rats. Simultaneously a raised level of ANF messenger RNA in morphine tolerant rat atria was observed. It is suggested that the biosynthesis and storage of ANF in atria be increased and its release from atria decreased in morphine tolerant rats.

[Construction of a cDNA library of Schistosoma japonicum].

The template mRNA was extracted from Schistosoma japonicum. The first strand of cDNA was synthesized by AMV-reverse transcriptase. The second strand cDNA was first digested by RNase H to remove mRNA and was then synthesized by AMV-reverse transcriptase, T4-DNA polymerase. Sizing of cDNA was applied on a NACS column to remove small fragments of less than 1 kb. Homopolymeric tailing of vector (pUC18) was done with dGTP and DNA terminal transferase and tailing of the cDNA with dCTP was carried out under the same conditions. After annealing, the plasmids with cDNA were transformed into E. coli MC1061. The efficiency of cloning was about 10(4)/micrograms mRNA with 30% of the transformants having the inserts of cDNA (Figs. 1-2).

[Screening of Schistosoma japonicum adult worm genomic DNA library by specific antibody].

S. japonicum adult worm genomic DNA libraries were screened by enzymeimmunoassay. The antigens produced by recombinant lambda gt 11 plaques were transferred to nitrocellulose filters. Clones encoding given antigens were detected with infected rabbit sera (IRS) which were preabsorbed with lysate of induced lambda gt 11 in Y1090 cells. Eight putative positives were picked up from 97 plates in the primary screening and were rescreened a second time, and, 2 of them consistently gave good positive signals in subsequent screenings. One of the phage clones was purified to homogeneity and mixed with Y1089 cells. The expressed products still showed positive when rescreened by ELISA, indicating that the clone encoding S. japonicum antigen could be recognized by IRS. The immunoscreening method described here was able to efficiently isolate single clones encoding S. japonicum antigens. The method has the advantages of screening libraries efficiently, reliably and specifically, and it might open the way to screen genes encoding S. japonicum antigens inducing protective immunity (Fig. 1).

A prototype recombinant vaccine against respiratory syncytial virus and parainfluenza virus type 3.

We have produced a genetically-engineered chimeric protein composed of the external domains of the respiratory syncytial virus (RSV) fusion (F) protein and the parainfluenza virus type 3 (PIV-3) hemagglutinin-neuraminidase (HN) protein in insect cells using the baculovirus expression system. The yield of the soluble chimeric FRSV-HNPIV-3 protein could be increased approximately 2-fold by using Trichoplasia ni (High Five) insect cells in place of Spodoptera frugiperda (Sf9) for expression. The chimeric protein, purified from the supernatant of baculovirus-infected High Five cells by immunoaffinity chromatography was correctly processed at the F2-F1 proteolytic cleavage site. Immunochemical analysis of the chimera with a panel of anti-F and anti-HN monoclonal antibodies suggested that the antigenicity of the major F and HN neutralization epitopes of the chimeric protein was preserved. Immunization of cotton rats with two 1 or 10 micrograms doses of the chimeric protein adsorbed to aluminum phosphate elicited strong PIV-3 specific HAI responses as well as PIV-3 and RSV specific neutralizing antibodies, and at either dose completely protected against challenge with live RSV and PIV-3.

Physical and biological significance of peptide sequences mediating the interaction between high molecular weight kininogen and plasma prekallikrein.

HK31 (S565-K595) has previously been shown to encompass the binding domain for plasma prekallikrein (PK) within domain 6 of high molecular weight kininogen (HK). The complementary binding domain for HK within PK is mapped to PK56 (F56-G86), in the Apple 1 domain and to PK266 (K266-C295) in the Apple 4 domain. Isothermal titration calorimetry demonstrated that either PK peptide binds to HK31 in 1:1 stoichiometry. Binding of the alternate PK peptide into a ternary complex is facilitated nearly 2-fold. Fluorescence emission spectroscopy revealed that only the binding of PK56 caused a limited decrease in intrinsic tryptophane fluorescence emission intensity of HK31. We conclude that the two PK peptides bind to the HK peptide at different sites. To map the minimal sequence within HK31, truncated new peptides were tested for their ability to compete with HK for binding PK in a cell-free system. D567-T591, a 25-residue peptide which contains sufficient structural information for binding kallikrein in solution, blocked the binding of kallikrein to HK bound to endothelial cells and inhibited PK activation to kallikrein and the generation of kallikrein-activated urokinase on endothelial cell surfaces. HK-derived peptides could modulate excessive fibrinolysis and hypotension in sepsis and multiple trauma.

[Study on the DNA vaccine against foot-and-mouth disease virus using the heavy chain constant region of swine IgG as the carrier for peptide epitopes].

The peptide of amino acids 141-160 of VP1 protein of foot-and-mouth disease virus (FMDV) is a major B cell epitope and the peptide of amino acids 21-40 is an important T cell epitope. In this study, the DNA fragments of 141-160 and 21-40 peptide epitopes of a strain of type O FMDV was chemically synthesized and arranged into a tandem repeat 141-160 (20AA)-21-40 (20AA)-141-160 (20AA). This tandem sequence was fused to the 3' end of the heavy chain constant region gene of swine immunoglobulin G and was then cloned into mammalian expression vector pCDM8 to form a recombinant plasmid pCDM8FZ3. After pCDM8FZ3 was inoculated intramuscularly into guinea pigs, it elicited a neutralizing antibody response and a specific spleen T cell proliferative response, and 66% of the vaccinated animals were protected from viral challenge. Our study indicated that the heavy chain constant region of swine IgG can act as the carrier protein for FMDV peptide epitopes, and pC-DM8FZ3 is a potential DNA vaccine candidate to prevent FMDV infection.

Baculovirus expression of the respiratory syncytial virus fusion protein using Trichoplusia ni insect cells.

Respiratory syncytial virus (RSV) is a major viral pathogen responsible for severe respiratory tract infections in infants, young children, and the elderly. The RSV fusion (F) protein is highly conserved among RSV subgroups A and B and is the major protective immunogen. A genetically-engineered version of the RSV F protein was produced in insect cells using the baculovirus expression system. To express a secreted form of this protein, the transmembrane domain was eliminated by removing the region of the gene encoding 48 amino acids at the C-terminus. Production of the truncated RSV F protein (RSV-Fs) was compared in two different insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five). The yield of RSV-Fs secreted from High Five insect cells was over 7-fold higher than that from Sf9 insect cells. Processing of the RSV-Fs protein was also different in the two insect cell lines. N-terminal sequencing demonstrated that while most of the RSV-Fs protein secreted by High Five cells was correctly processed at the F2-F1 proteolytic cleavage site, most of the RSV-Fs protein secreted by Sf9 cells was unprocessed or incorrectly processed. Antigenicity of the major RSV F neutralization epitopes was maintained in the RSV-Fs protein secreted from High Five cells. The RSV-specific neutralizing antibody titres in the sera of cotton rats immunized with the RSV-Fs protein were equivalent to those in the sera of animals intranasally inoculated with live RSV. Animals immunized with either live RSV or the immunoaffinity purified RSV-Fs protein from High Five cells were completely protected against live virus challenge.

Plasmids encoding foot-and-mouth disease virus VP1 epitopes elicited immune responses in mice and swine and protected swine against viral infection.

VP1 is a capsid protein of foot-and-mouth disease virus (FMDV) and contains epitopes of the virus. Plasmids encoding two VP1 epitopes (amino acid residues 141-160 and 200-213) and a host-self immunoglobulin molecule were constructed to produce a new type of FMD DNA vaccine. Two plasmids, namely, pCEIM and pCEIS, containing mouse immunoglobulin (IgG) or swine IgG were subjected to immunogenicity testing in mice and swine, respectively. In mice administrated pCEIM in the abdomen using a genegun, both FMDV-specific T-cell proliferation and neutralizing antibodies were detected. In swine immunized with pCEIS at the back of the ear, immune responses were achieved after the second administration. Swine showed a T-cell proliferative response with a stimulation index (SI) of up to 8.1 and a neutralizing antibody response that was able to protect suckling mice from 10(2) LD(50) (lethal dose 50) FMDV challenge. To compare the immunogenicity of the DNA-based vaccine candidate, versus the protein-based vaccine candidates, a second group of swine was immunized with the protein F1-scIgG, which was encoded by the plasmid pCEIS. Injection with F1-scIgG elicited a T-cell proliferative response of SI < 1.7 and a neutralizing antibody response that protected suckling mice from up to 10(5) LD(50) FMDV challenge. In the challenge test, three of three swine immunized with pCEIS were fully protected from FMDV challenge.