Objective:To investigate the levels of diphtheria-specific binding antibodies and neutralizing antibodies in mice immunized with pneumococcal polysaccharide conjugate vaccine using diphtheria toxoid as a carrier.Methods:NIH mice were immunized with one batch of diphtheria, tetanus and acellular pertussis combined vaccine, absorbed (DTaP-1) or three different batches of 13-valent pneumococcal polysaccharide conjugate vaccine (PCV13, containing diphtheria toxoid vector) at three dilutions (5-, 10- and 20-fold dilution). Serum samples were collected to test for diphtheria-specific antibody titers and diphtheria potencies of the vaccines. Another three batches of DTaP vaccine and three batches of tetanus toxoid, reduced diphtheria toxoid and acellular pertussis combined vaccine (Tdap) were used to immunize NIH mice. Serum samples were collected and the diphtheria potencies were detected. Statistical analysis was performed using one-way analysis of variance.Results:At the 5-fold and 10-fold dilutions, the titers of diphtheria-specific antibodies induced by three batches of PCV13 vaccine were all lower than those by DTaP-1 vaccine (all P<0.001), while there was no statistically significant difference at the 20-fold dilution ( P>0.05). The diphtheria potencies of the DTaP-1 vaccine and the three batches of PCV13 vaccine were 100.5, 76.2, 64.5, and 62.0 IU/ml, respectively. The diphtheria potencies of another three batches of DTaP vaccine were 82.5, 83.6, and 79.9 IU/ml, respectively, and those of three batches of Tdap vaccine were 10.3, 12.2, and 12.9 IU/ml, respectively. There was no statistically significant difference in diphtheria potency between DTaP vaccine and PCV13 vaccine( P>0.05), while there was a statistically significant difference between Tdap vaccine and the PCV13 vaccine ( P<0.001). Conclusions:The pneumococcal polysaccharide conjugate vaccine with diphtheria toxoid has good diphtheria immunogenicity and can induce the production of higher levels of diphtheria-specific binding antibodies and protective neutralizing antibodies in vivo. Pneumococcal capsular polysaccharide exerts an immune enhancement effect on diphtheria toxoid. The relevant results provide valuable guidance for determining carrier protein dosage in bacterial polysaccharide conjugate vaccines, planning vaccine co-administration, and selecting the dosage of diphtheria toxoid antigen in the research and development of combined vaccines.

Objective:To investigate the levels of diphtheria-specific binding antibodies and neutralizing antibodies in mice immunized with pneumococcal polysaccharide conjugate vaccine using diphtheria toxoid as a carrier.Methods:NIH mice were immunized with one batch of diphtheria, tetanus and acellular pertussis combined vaccine, absorbed (DTaP-1) or three different batches of 13-valent pneumococcal polysaccharide conjugate vaccine (PCV13, containing diphtheria toxoid vector) at three dilutions (5-, 10- and 20-fold dilution). Serum samples were collected to test for diphtheria-specific antibody titers and diphtheria potencies of the vaccines. Another three batches of DTaP vaccine and three batches of tetanus toxoid, reduced diphtheria toxoid and acellular pertussis combined vaccine (Tdap) were used to immunize NIH mice. Serum samples were collected and the diphtheria potencies were detected. Statistical analysis was performed using one-way analysis of variance.Results:At the 5-fold and 10-fold dilutions, the titers of diphtheria-specific antibodies induced by three batches of PCV13 vaccine were all lower than those by DTaP-1 vaccine (all P<0.001), while there was no statistically significant difference at the 20-fold dilution ( P>0.05). The diphtheria potencies of the DTaP-1 vaccine and the three batches of PCV13 vaccine were 100.5, 76.2, 64.5, and 62.0 IU/ml, respectively. The diphtheria potencies of another three batches of DTaP vaccine were 82.5, 83.6, and 79.9 IU/ml, respectively, and those of three batches of Tdap vaccine were 10.3, 12.2, and 12.9 IU/ml, respectively. There was no statistically significant difference in diphtheria potency between DTaP vaccine and PCV13 vaccine( P>0.05), while there was a statistically significant difference between Tdap vaccine and the PCV13 vaccine ( P<0.001). Conclusions:The pneumococcal polysaccharide conjugate vaccine with diphtheria toxoid has good diphtheria immunogenicity and can induce the production of higher levels of diphtheria-specific binding antibodies and protective neutralizing antibodies in vivo. Pneumococcal capsular polysaccharide exerts an immune enhancement effect on diphtheria toxoid. The relevant results provide valuable guidance for determining carrier protein dosage in bacterial polysaccharide conjugate vaccines, planning vaccine co-administration, and selecting the dosage of diphtheria toxoid antigen in the research and development of combined vaccines.

Objective: Analyzing the characteristics of consonant errors in children with functional dysarthria in different age groups and the effect of speech training provides a reference for clinical treatment. Methods : This study followed medical ethics, and informed consent has been obtained from patients. Speech data from 388 patients with functional dysarthria were retrospectively studied. They were divided into two groups at the age of 6, namely, the preschool group (4-6 years old) of 226 patients and the school age group (6-13 years old, including 6 years old) of 162 patients. The characteristics of consonant pronunciation errors from four aspects were analyzed: average number of errors, pronunciation location, pronunciation method, and error type. One-on-one speech training was conducted, with a training frequency of once a week and once for 30 minutes. The training method was carried out in the order of phoneme training, syllable training, vocabulary training, sentence training, and short text and conversation training. The effects of speech training in the two groups were compared. Results: Analysis by pronunciation location: both age groups had the highest frequency of errors in tongue tip posterior sounds; the school age group had the lowest error frequency for labiodental consonants, and the preschool group had the lowest error frequency for bilabial consonants. According to the analysis of pronunciation mode, both age groups had the highest error frequency of aspirated affricate and the lowest error frequency of nasal sound. Analysis by error type: both age groups are mainly characterized by substitution and omission. Compared with the preschool group, most consonants of patients in the school group tend to improve in terms of pronunciation location, pronunciation mode, and error types. Compared with the preschool group, the two types of errors-palatalization and lateralization-increased in frequency in the school group, but the trend of increased lateralization was not statistically significant. After 6.7 and 5.5 sessions of speech training, the pronunciation of the preschool group and the school-age group significantly improved; the cure rate of the school-age group was 84.9% (118/139), and that of the preschool group was 77.1% (91/118). There was no statistically significant difference in the cure rate between the two groups. Conclusion Functional dysarthria may improve with age, but it may not completely self-heal. Children of different age groups can achieve good treatment results through scientific and reasonable speech training.

Flavanone 3-hydroxylase (F3H) is a key enzyme in the synthesis of phycocyanidins. In this experiment, the petals of red Rhododendron hybridum Hort. at different developmental stages were used as experimental materials. The R. hybridum flavanone 3-hydroxylase (RhF3H) gene was cloned using reverse transcription PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques, and bioinformatics analyses were performed. Petal RhF3H gene expression at different developmental stages were analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR). A pET-28a-RhF3H prokaryotic expression vector was constructed for the preparation and purification of RhF3H protein. A pCAMBIA1302-RhF3H overexpression vector was constructed for genetic transformation in Arabidopsis thaliana by Agrobacterium-mediated method. The results showed that the R. hybridum Hort. RhF3H gene is 1 245 bp long, with an open reading frame of 1 092 bp, encoding 363 amino acids. It contains a Fe²⁺ binding motif and a 2-ketoglutarate binding motif of the dioxygenase superfamily. Phylogenetic analysis showed that the R. hybridum RhF3H protein is most closely related to the Vaccinium corymbosum F3H protein. qRT-PCR analysis showed that the expression level of the red R. hybridum RhF3H gene tended to increase and then decrease in the petals at different developmental stages, with the highest expression at middle opening stage. The results of the prokaryotic expression showed that the size of the induced protein of the constructed prokaryotic expression vector pET-28a-RhF3H was about 40 kDa, which was similar to the theoretical value. Transgenic RhF3H Arabidopsis thaliana plants were successfully obtained, and PCR identification and β-glucuronidase (GUS) staining demonstrated that the RhF3H gene was integrated into the genome of A. thaliana plants. qRT-PCR, total flavonoid and anthocyanin contentanalysis showed that RhF3H was significantly higher expressed in the transgenic A. thaliana relative to that of the wild type, and its total flavonoid and anthocyanin content were significantly increased. This study provides a theoretical basis for investigating the function of RhF3H gene, as well as for studying the molecular mechanism of flower color in R. simsiib Planch.
Arabidopsis/metabolism* ; Rhododendron/metabolism* ; Amino Acid Sequence ; Anthocyanins/metabolism* ; Phylogeny ; Flavonoids/metabolism* ; Cloning, Molecular ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism*

Terpene synthase (TPS) plays important roles in the synthesis of terpenoids which are the main fragrances in Rhododendron flowers. To understand the function of TPS genes in terpenoid metabolism in relation to flower aroma formation, we identified all TPS gene family members in Rhododendron by analyzing its genome database. We then used a transcriptomic approach to analyze the differential gene expression patterns of TPS gene family members in the scented flower Rhododendron fortunei compared to the non-scented flower Rhododendron 'Nova Zembla'. The contents of terpenoid compounds in petals of the above two Rhododendron species at different developmental stages were also measured by using qRT-PCR and head space-solid phase micro-extraction combined with gas chromatography-mass spectrometry. Our results showed that a total of 47 RsTPS members, with individual lengths ranged from 591 to 2 634 bp, were identified in the Rhododendron genome. The number of exons in RsTPS gene ranged from 3 to 12, while the length of each protein encoded ranged from 196 to 877 amino acids. Members of the RsTPS family are mainly distributed in the chloroplast and cytoplasm. Phylogenetic analysis showed that RsTPS genes can be clustered into 5 subgroups. Seven gene family members can be functionally annotated as TPS gene family since they were temporally and spatially expressed as shown in the transcriptome data. Notably, TPS1, TPS10, TPS12 and TPS13 in Rhododendron fortunei were expressed highly in flower buds reached the peak in the full blossoming. Correlation analysis between gene expression levels and terpenoid content indicates that the expression levels of TPS1, TPS4, TPS9, TPS10, TPS12 and TPS13 were positively correlated with the content of terpenoids in the petals of R. fortunei at all flower developmental stages, suggesting that these six genes might be involved in the aroma formation in R. fortunei.
Rhododendron/metabolism* ; Phylogeny ; Terpenes/metabolism* ; Family ; Gene Expression Regulation, Plant

Background Under the background of global climate change, temperature has increased dramatically. Most studies about association between temperature and human health are conducted in low-altitude areas, but rarely focus on plateau areas. Objective To examine the association between temperature and non-accidental mortality risk in Tibet Plateau, China and to identify vulnerable populations for formulating targeted policies of climate change adaptation. Methods The mortality data, meteorological data, and pollutant data of Tibet area between 2013 to 2019 were collected. Based on time-stratified case-crossover design, conditional logistic regression models were used to analyze the exposure-response relationship between temperature and cause-specific mortality, which was linearized to obtain excess risk for 1 ℃ change; attributable fraction was calculated for assessing burden attributable to temperature; and stratified analyses were further conducted by gender, age (＜65 years old, ≥65 years old), and causes of death (cardiovascular diseases, cerebrovascular diseases, and respiratory diseases). Sensitivity analyses were conducted by adjusting model parameters and variables. Results A total of 26 045 non-accidental deaths were collected in Tibet during 2013 and 2019, and the P₅₀ of temperature was 5.0 ℃. The non-accidental mortality risk increased as temperature become colder. A 1 ℃ decrease in temperature was associated with a 2.01% (95%CI: 0.94%-3.07%) increase in total non-accidental mortality, while the association changed to 2.05% (95%CI: 0.62%-3.47%) for male and 1.96% (95%CI: 0.34%-3.56%) for female, both of statistial significance; 1.45% (95%CI: −0.10%-2.98%) for the people <65 years old (not of significance) and 2.52% (95% CI : 1.04%-3.99%) for the people ≥65 years old (of significance); the excess risk for cardiovascular mortality was 2.65% (95%CI: 1.03%-4.24%), for cerebrovascular mortality was 3.70% (95%CI: 0.74%-6.57%), both of statistical significance, and for respiratory mortality was 2.18% (95%CI: −0.14%-4.44%), without significance. The total attribution number of non-accidental mortality was 5340 (95%CI: 2719-7528), and the total attributable fraction was 20.50% (95%CI: 10.44%-28.91%). The attributable fractions were higher in specific subgroups like male (20.72%), people ≥65 years (23.33%), and people with cardiovascular diseases (26.07%). Conclusion The exposure-response relationship between temperature and non-accidental mortality in Tibet showes that the non-accidental mortality risk increase as temperature become colder. The attributable burden of disease is heavy. Residents being male, ≥65 years, with cardiovascular diseases and respiratory diseases may be vulnerable to nonoptimal temperature.

Phenylalaninammo-nialyase (PAL) is a key enzyme in the synthesis of methyl benzoate - a plant aroma compound. In order to understand the function of this enzyme in the formation of fragrance in the scented Rhododendron species-Rhododendron fortunei, we cloned a gene encoding this enzyme and subsequently examined the gene expression patterns and the profile of enzyme activity during development in various tissues. The full length of RhPAL gene was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. The expression levels of RhPAL gene were measured by real-time quantitative reverse transcription PCR (qRT-PCR) and the amount of phenylalanine and cinnamic acid were assayed with LC-MS. The results showed that the ORF sequence of RhPAL gene amplified from the cDNA templates of flower buds had 2 145 bp, encoding 715 amino acids, and shared 90% homology to the PAL amino acid sequences from other species. qRT-PCR analysis showed that the expression of RhPAL in petals during flowering kept in rising even until the flowers wilted. The expression of RhPAL in pistil was much higher than that in stamen, while the expression in the younger leaves was higher than in old leaves. However, the expression level was relatively lower in petal and stamen compared to that in leaves. We also measured the PAL activity by Enzyme-linked immuno sorbent assay in the petals of flowers at different flowering stages. The results showed that PAL activity reached the highest at the bud stage and then decreased gradually to the lowest when the flowers wilted, which followed a similar trend in the emission of the flower fragrance. The phenylalanine and cinnamic acid contents measured by LC-MS were highly correlated to the expression level of RhPAL in various tissues and at different flowering stages, implying that RhPAL plays an important role in the formation of the flower fragrance. This work may facilitate the breeding and improvement of new fragrant Rhododendron cultivars.
Amino Acid Sequence ; Cloning, Molecular ; DNA, Complementary ; Flowers/genetics* ; Rhododendron/genetics*

OBJECTIVE: To analyze the clinical features, biochemical characteristics and molecular pathogenesis of a girl with isovaleric acidemia. METHODS: Clinical features, blood spot amino acid profiles and urinary organic acid profiles of the patient were analyzed. Targeted capture, next generation sequencing and Sanger sequencing were carried out to detect potential variant of the IVD gene. RESULTS: The patient presented with poor weight gain, poor feeding, lethargy, and a "sweaty feet" odor 10 days after birth. Biochemical test suggested hyperammonemia. Blood spot amino acid profiles displayed a dramatic increase in isovalerylcarnitine (C5: 3. 044, reference range 0.04 - 0.4 μmol/L). Organic acid analysis of her urine sample revealed a high level of isovaleric glycine (669. 53, reference range 0 - 0.5). The child was ultimately diagnosed with isovaleric acidemia, and was found to harbor a paternally derived heterozygous variant c.149G>A (p.R50H) and a maternally derived heterozygous variant c.1123G>A (p.G375S) of the IVD gene. Her elder brother was a heterozygous carrier of c.1123G>A (p.G375S) variant. The c.149G>A (p.R50H) was a known pathogenic variant, while the c.1123G>A (p.G375S) variant was previously unreported. CONCLUSION The pathogenesis of the patient was delineated from the perspective of genetics, which has provided a basis for clinical diagnosis, treatment as well as genetic counseling.
Amino Acid Metabolism, Inborn Errors/genetics* ; Child ; Female ; Heterozygote ; Humans ; Isovaleryl-CoA Dehydrogenase/genetics* ; Male ; Mutation

Objective:To provide clinical references for the diagnosis and treatment of hemophilic pseudotumor (HPT) in maxillofacial region.Methods:Fourteen cases of HPT in maxillofacial region from the Department of Stomatology, Beijing Children′s Hospital from Jan 2009 to Jan 2019 were collected. Two cases were lost for follow-up and 12 patient,all boys, were finally followed up and included in the study. The patients aged from 13 months to 10 years old. The medical history, clinic manefestitions and the features of the radiology examination were recorded. The patients were treated by using replacement treatment first. If the conservative treatment was not effective, the patients then received operation combined with pereoperation replacement thearapy. The patients were followed up for 13 months to 10 years.There were 11 cases of hemophilia A, and 1 case of hemophilia B. Two cases were severe type, the others (10/12) were mild and moderate types. Only 1 case was diagnosed as hemophilia initially. Nine cases (9/12) were misdiagnosed as malignant tumors, 1 case was misdiagnosed as osteomyelitis and 1 case was misdiagnosed as hemangioma. Only 3 cases had identified history of trauma before.Results:All cases were treated with replacement therapy first, among which 10 cases were effective, 8 cases were cured by conservative therapy, 1 case had residual soft tissue fistula after conservative treatment and 1 case recurrented after conservative treatment for 8 months. Two patients with poor efficacy to the replacement treatment were performed operations and finally were cured.Conclusions:The misdiagnosis rate of HPT in maxillofacial region was high. The conservative factor replacement therapy could achieve good results in most children and could be used as the preferred treatment. If the conservative treatment was not effective, the surgical treatment was also a safe option.

Objective To establish a method for simultaneous determination for the contents of four flavones (ononin, calycosin, genistein and formononetin) of Hedysari Radix in Gansu Province with quantitative analysis of multi-components by single-marker (QAMS); To prove its feasibility and accuracy. Methods Calycosin was taken as internal standard substance. Relative correction factors (RCF) of ononin, genistein and formononetin to calycosin were established. The contents of ononin, calycosin, genistein and formononetin were determined to realize QAMS. Results RCF was with good repeatability. The results of QAMS were consistent with the results of the external standard method. Conclusion The method that determines the contents of ononin, genistein and formononetin with calycosin as internal standard substance, can be used for quantitative analysis of Hedysari Radix.