Objective Currently, there is no commercially available quantitative detection kit for the main Felis domestic allergen (Fel d 1) in China. To establish a rapid detection method for Fel d 1, this study aims to prepare monoclonal antibodies against Fel d 1 protein. Methods The codon preference of Escherichia coli was utilized to optimize and synthesize the Fel d 1 gene. The prokaryotic expression plasmid pET-28a-Fel d 1 was constructed and used to express and purify the recombinant Fel d 1 protein. Subsequently, the recombinant protein was immunized into BALB/c mice and monoclonal antibodies (mAbs) were prepared by the hybridoma technique. An indirect ELISA was established using the recombinant Fel d 1 as the coating antigen, and hybridoma cell lines were screened for positive clones. The specificity and antigenic epitopes of the mAbs were confirmed by Western blot analysis. Finally, the selected hybridoma cells were injected into the peritoneal cavities of BALB/c mice for large-scale monoclonal antibody production. Results The recombinant plasmid pET-28a-Fel d 1 was successfully constructed, and soluble Fel d 1 protein was obtained after optimizing the expression conditions. Western blot and antibody titer assays confirmed the successful isolation of two hybridoma cell lines, 7D11 and 5H4, which stably secreted mAbs specific to Fel d 1. Antibody characterization revealed that the 5H4 mAb was of the IgG2a subtype and could recognize the amino acid region 105-163 of Fel d 1, while the 7D11 mAb was the IgG1 subtype and could recognize the amino acid region 1-59. Conclusion The high-purity recombinant Fel d 1 protein produced in this study provides a promising alternative for clinical immunotherapy of cat allergies. Furthermore, the monoclonal antibody prepared in this experiment lays a material foundation for the in-depth study of the biological function of Fel d 1 and the development of ELISA detection.
Animals ; Antibodies, Monoclonal/biosynthesis* ; Mice, Inbred BALB C ; Cats ; Mice ; Allergens/genetics* ; Glycoproteins/genetics* ; Enzyme-Linked Immunosorbent Assay ; Hybridomas/immunology* ; Recombinant Proteins/genetics* ; Female ; Antibody Specificity

Objective·To study the relationship between evolution and the developmental process from the perspective of DNA sequence conservation,and explore their inherent principles.Methods·First,conservation rate(CR)was established by analyzing the conservation of amino acid sequences of coding genes in 100 species to quantify the evolutionary conservation of genes.The relationship between CR and developmental potential was verified by using the feature genes involved in embryonic stem cells pathways.Secondly,cell type-specific genes and their characteristics in conservation were studied by analyzing the RNA sequencing(RNA-seq)data of the three early germ layers(ectoderm,mesoderm and endoderm)and their corresponding mature organs(brain,heart,liver,etc).Then,chromatin immunoprecipitation sequencing(ChIP-seq)data of enhancer histone H3 acetylated at lysine 27(H3K27ac)from early germ layers and mature organs were collected to search for enhancer sites and identify super enhancers in various cells and tissues by using the ROSE procedure.Functional enrichment and signaling pathway analysis of genes was used to examine the identity correlation between SEs-regulated genes and the corresponding cell characteristics,to clarify whether the SEs identified in this study were consistent with the characteristics reported in previous studies.Finally,PhastCons program was used to calculate the DNA conservation score(CS)of non-coding regulatory regions to study their relationship with developmental potential.Results·In the coding region of DNA,CR was successfully established to quantify the conservation of genes.The gene expression data of early germ layers and mature organs showed that the genes with higher conservation rate were more relevant to the stemness and early developmental process,and the differences between the tissues from early and late development could be distinguished by using CR.In the non-coding regions of DNA,it was found that the conservation of regulatory regions was also correlated with development.The CS of the SE sequences in the early developmental germ layers was significantly higher than that of the SE sequences in the corresponding mature organs.However,cell-specific typical enhancers(TEs)did not show such a trend.Conclusion·During the developmental process,CR of genes expressed in the coding region decreases,and CS of super-enhancer DNA in the non-coding region decreases.

The importance of structural variants(SVs)for human phenotypes and diseases is now recognized.Although a variety of SV detection platforms and strategies that vary in sensitivity and specificity have been developed,few benchmarking procedures are available to confidently assess their performances in biological and clinical research.To facilitate the validation and application of these SV detection approaches,we established an Asian reference material by characterizing the genome of an Epstein-Barr virus(EBV)-immortalized B lymphocyte line along with identified benchmark regions and high-confidence SV calls.We established a high-confidence SV callset with 8938 SVs by integrating four alignment-based SV callers,including 109x Pacific Biosciences(PacBio)continuous long reads(CLRs),22 x PacBio circular consensus sequencing(CCS)reads,104x Oxford Nanopore Technologies(ONT)long reads,and 114×Bionano optical mapping plat-form,and one de novo assembly-based SV caller using CCS reads.A total of 544 randomly selected SVs were validated by PCR amplification and Sanger sequencing,demonstrating the robustness of our SV calls.Combining trio-binning-based haplotype assemblies,we established an SV benchmark for identifying false negatives and false positives by constructing the continuous high-confidence regions(CHCRs),which covered 1.46 gigabase pairs(Gb)and 6882 SVs supported by at least one diploid haplotype assembly.Establishing high-confidence SV calls for a benchmark sample that has been characterized by multiple technologies provides a valuable resource for investigating SVs in human biology,disease,and clinical research.

Objective:To analyze the clinical effect of hyaluronidase injection through the facial artery in the treatment of vascular embolism such as skin ulceration and necrosis after cosmetic injection.Methods:Hyaluronidase was injected through facial artery in 13 patients who were diagnosed with vascular embolism after facial injection from January 2019 to May 2020. The facial artery was punctured with 22-gauge arterial blood collection needle or 19/23-gauge disposable venous infusion needle. The angle between the needle body and the skin varies depending on the patients’ weight, ranged 30°-45°. The needle was advanced slowly and pushed forward by 2-3 mm when blood backflow appeared in the needle core. After confirming the successful puncture of the facial artery, 0.5-1.5 ml hyaluronidase was slowly injected into the facial artery. The time of skin relaxation, tenderness relief, ulcer healing and wound recovery were observed. The pigmentation was observed and the Vancouver Scar Scale (VSS) was used to score the scars after 3-12 months.Results:A toal of 13 patients with vascular complications of hyaluronidase filler were retrospectively reviewed. The patients were 18-45-year-old(mean age, 35 years) and received hyaluronidase filler at private clinics. There were 12 women and 1 man. The time from onset to visit was 14 h to 4 d, with an average time of 2.5 d. Hyaluronidase was most commonly injected into the nasolabial folds (54%, 7 of 13). The second-ranked area is the nasalroot (23%, 3 of 13). These patients had skin swelling, necrosis, ecchymosis or black scabs during or after hyaluronidase injection. Some patients showed skin lesions combined with oral ulcer. After percutaneous facial arterial hyaluronidase injection, the local skin tissue injuries of the 13 patients were improved in time. The time of skin relaxation was (0.77±0.25) d, the time of tenderness relief was (1.23±0.64) d, the time of ulcer healing was (3.14±0.64) d and the time of wound recovery was (5.85±0.86) d. Patients were followed up for 3-12 months, with an average of 7 months. One patient had slight scar (VSS score of 1), two patients had only mild pigmentation (VSS score of 0), and the other ten patients had no scar and pigmentation (VSS score of 0).Conclusions:It is effective to improve local microcirculation and reduce skin tissue injury after percutaneous facial artery hyaluronidase injection in the treatment of skin injury caused by facial filler injection.

Objective:To analyze the clinical effect of hyaluronidase injection through the facial artery in the treatment of vascular embolism such as skin ulceration and necrosis after cosmetic injection.Methods:Hyaluronidase was injected through facial artery in 13 patients who were diagnosed with vascular embolism after facial injection from January 2019 to May 2020. The facial artery was punctured with 22-gauge arterial blood collection needle or 19/23-gauge disposable venous infusion needle. The angle between the needle body and the skin varies depending on the patients’ weight, ranged 30°-45°. The needle was advanced slowly and pushed forward by 2-3 mm when blood backflow appeared in the needle core. After confirming the successful puncture of the facial artery, 0.5-1.5 ml hyaluronidase was slowly injected into the facial artery. The time of skin relaxation, tenderness relief, ulcer healing and wound recovery were observed. The pigmentation was observed and the Vancouver Scar Scale (VSS) was used to score the scars after 3-12 months.Results:A toal of 13 patients with vascular complications of hyaluronidase filler were retrospectively reviewed. The patients were 18-45-year-old(mean age, 35 years) and received hyaluronidase filler at private clinics. There were 12 women and 1 man. The time from onset to visit was 14 h to 4 d, with an average time of 2.5 d. Hyaluronidase was most commonly injected into the nasolabial folds (54%, 7 of 13). The second-ranked area is the nasalroot (23%, 3 of 13). These patients had skin swelling, necrosis, ecchymosis or black scabs during or after hyaluronidase injection. Some patients showed skin lesions combined with oral ulcer. After percutaneous facial arterial hyaluronidase injection, the local skin tissue injuries of the 13 patients were improved in time. The time of skin relaxation was (0.77±0.25) d, the time of tenderness relief was (1.23±0.64) d, the time of ulcer healing was (3.14±0.64) d and the time of wound recovery was (5.85±0.86) d. Patients were followed up for 3-12 months, with an average of 7 months. One patient had slight scar (VSS score of 1), two patients had only mild pigmentation (VSS score of 0), and the other ten patients had no scar and pigmentation (VSS score of 0).Conclusions:It is effective to improve local microcirculation and reduce skin tissue injury after percutaneous facial artery hyaluronidase injection in the treatment of skin injury caused by facial filler injection.

OBJECTIVE: To explore the genetic basis for a fetus with autosomal recessive polycystic kidney disease (ARPKD). METHODS: Fetal tissue and peripheral blood samples were respectively obtained from the abortus and the couple. Following extraction of genomic DNA, genetic testing was carried out. RESULTS: The fetus was found to carry compound heterozygous variants of the PKHD1 gene, namely c.5336A>T (p.N1779I) and c.9455delA (p.N3152Tfs*10), which were respectively inherited from the husband and wife. CONCLUSION The c.5336A>T and c.9455delA variants of the PKHD1 gene probably account for the ARPKD in the fetus. Above results have enabled genetic counseling and prenatal diagnosis for the couple.

Objective:To screen and identify differentially expressed long non-coding RNA (lncRNAs) in peripheral blood of children with sepsis, and to explore the role of lncRNAs in the pathogenesis of sepsis in children.Methods:The peripheral blood samples of 3 children with sepsis admitted to the ICU of Children′s Hospital of Capital Institute of Pediatrics from January to December 2016 and 3 healthy children who underwent physical examination in our hospital during the same period were selected, and the differential expression profiles of lncRNAs and mRNAs were screened by lncRNAs sequencing technology.The target genes of differentially expressed lncRNAs were predicted and the relationship pairs of lncRNA-mRNA related to F 1F O-ATPase activity were constructed according to the results of GO analysis.Further increasing the sample size, we verified the expression of some F 1F O-ATPase activity-related mRNAs and lncRNAs which were differentially expressed in the screening results by real-time fluorescent quantitative polymerase chain reaction(qRT-PCR). Results:Sequencing results showed that there were 252 lncRNAs with significant differential expression in peripheral blood of children with sepsis compared with healthy children, of which 86 were up-regulated and 166 were down-regulated; meanwhile, there were 2 652 mRNAs with significant differential expression, of which 955 were up-regulated and 1 697 were down-regulated.The results of qRT-PCR showed that the expression of lncRNA ENST00000621933.1, ENST00000616950.1 and ENST00000595748.1 in peripheral blood of children with sepsis increased( P<0.05), while the expression of MT-ATP8, ATP5E and ENST00000624705.1, ENST00000615535.1 in peripheral blood of children with sepsis decreased( P<0.05), which was consistent with the sequencing results. Conclusion:lncRNAs are differentially expressed in peripheral blood of children with sepsis compared with healthy children.The expression levels of lncRNA ENST00000621933.1, ENST00000616950.1, ENST00000595748.1, ENST00000624705.1 and ENST00000615535.1 which their target genes are MT-ATP8 and ATP5E may be related to the development of sepsis in children.

Objective:To understand the epidemiological characteristics of infectious diarrhea pathogens in Pudong New Areas of Shanghai from 2013 to 2017 to provide evidence for control and prevention of the disease.Methods:From Jan 2013 to Dec 2017, active surveillance program on diarrhea was conducted in 14 sentinel hospitals (three tertiary-level and nine secondary-level, and two primary-level hospitals) in Pudong New Areas of Shanghai, based on location, catchment areas and number of patients. All recruited outpatients were interviewed in hospitals, using a standard questionnaire. Stool specimens were collected and tested for five viral and eight bacterial pathogens.Results:A total of 9 301 cases with infectious diarrhea were included, and the overall positive rate was 55.7 % (5 179). Positive rates of single virus, single bacteria and mixed infections were 26.7 % (2 481), 17.0 % (1 579) and 12.0 % (1 119), respectively. For single infection, the most commonly detected viruses appeared as norovirus (15.4 %, 1 428/9 301) and rotavirus (7.2 %, 667/9 301). The most commonly detected bacteria were diarrheagenic Escherichia coli (6.7 %, 619/9 301) and non-typhoid Salmonella (3.3 %, 305/9 301). The most common mixed infections were caused by virus-bacteria (4.9 %, 459/9 301). Norovirus (17.0 %, 838/4 938) showed the highest positive rates, followed by Escherichia coli (7.2 %, 354/4 938), both seen in the age group of 20-59 years old group. Rotavirus (9.4 %, 178/1 896) and non-typhoid Salmonella (4.9 %, 93/1 896) were the most common pathogens found in the age group of 0-4 years old. The prevalence of norovirus peaked both in spring and autumn. The other peaks were seen as: Rotavirus in winter, diarrheagenic Escherichia coli in summer and non-typhoid Salmonella in summer. Conclusions:Our data showed that the positive rates of infectious diarrhea pathogens were high in Pudong New Areas of Shanghai from 2013 to 2017. The dominant pathogens would include norovirus, rotavirus and diarrheagenic Escherichia coli but with differenct distributions in age groups. Obvious seasonal patterns were also observed.

PURPOSE: The significance of neuroendocrine differentiation (NED) in gastric carcinoma (GC) is controversial, leading to ambiguous concepts in traditional classifications. This study aimed to determine the prognostic threshold of meaningful NED in GC and clarify its unclear features in existing classifications. MATERIALS AND METHODS: Immunohistochemical staining for synaptophysin, chromogranin A, and neural cell adhesion molecule was performed for 945 GC specimens. Survival analysis was performed using the log-rank test and univariate/multivariate models with percentages of NED (PNED) and demographic and clinicopathological parameters. RESULTS: In total, 275 (29.1%) cases were immunoreactive to at least 1 neuroendocrine (NE) marker. GC-NED was more common in the upper third of the stomach. PNED, and Borrmann's classification and tumor, lymph node, metastasis stages were independent prognostic factors. The cutoff PNED was 10%, beyond which patients had significantly worse outcomes, although the risk did not increase with higher PNED. Tumors with ≥10% NED tended to manifest as Borrmann type III lesion with mixed/diffuse morphology and poorer histological differentiation; the NE components in this population mainly grew in insulae/nests, which differed from the predominant growth pattern (glandular/acinar) in GC with <10% NED. CONCLUSIONS: GC with ≥10% NED should be classified as a distinct subtype because of its worse prognosis, and more attention should be paid to the necessity of additional therapeutics for NE components.
Adenocarcinoma ; Chromogranin A ; Classification ; Humans ; Immunohistochemistry ; Lymph Nodes ; Neoplasm Metastasis ; Neural Cell Adhesion Molecules ; Prognosis ; Stomach ; Stomach Neoplasms ; Synaptophysin

Objective: To investigate the antimicrobial resistance and molecular epidemiology of foodborne Yersinia (Y.) enterocolitica in Pudong New District of Shanghai. Methods: Four kinds of raw food samples were collected in retail circulation sites in Pudong from 2012 to 2016. Cold enrichment method was used to isolate Y. enterocolitica and further detection of biotype, serotype, virulent genes, antimicrobial susceptibility of the isolates and pulsed field gel electrophoresis (PFGE) were conducted. Results: A total of 3 900 raw food samples were collected during this period, including poultry product (n=590), livestock product (n=1 074), aquatic product (n=1 488), vegetable (n=748), in which 111 (2.8%) were contaminated by Y. enterocolitica. The detection rates of Y. enterocolitica in poultry product samples (5.3%, 31/590) and livestock product samples (4.5%, 48/1 074) were higher than those in aquatic product samples (1.6%, 24/1 488) and vegetable samples (1.1%, 8/748). The predominant biotype was 1A (95.5%) and predominant serotype was O∶8 (42.3%). All the strains lacked ail, ystA, yadA and virF genes, which encoded pathogenic Y. enterocolitica. Seventy six (68.5%) strains harbored ystB gene, in which 35 (31.5%) belonged to 1A/O∶8/ystB pattern. Most strains were resistant to ampicillin (74.8%) and amoxicillin/clavulanic acid (70.3%), and non-sensitive rate to Cefoxitin was over 50.0%. No third generation cephalosporin or fluoroquinolone resistant strains were detected, but 38.7% (43/111) strains were multidrug resistant (MDR). Serotype O∶8 and O∶5 strains had 44 and 18 PFGE patterns, respectively. Conclusions The main foodborne exposure sources of Y. enterocolitica in raw food were poultry and livestock products in Pudong New District. 1A/O∶8/ystB was the predominant pattern with potential pathogenicity despite lacks of typical pathogenic virulent genes. The antimicrobial resistant rates of Y. enterocolitica were at a low level, but MDR strains still existed. Molecular types of the isolates showed highly genetic diversity.