To isolate human phage single chain antibody against surface protein of Streptococcus mutans,the recombinant surface protein of S.mutans(rAP) was used to coat the immune tubes and the phage single chain antibody was prepared through pDAN5 phage antibody library after 5 rounds of panning.The eluted phage was enriched nearly 30 times.In these ways,13 positive clones were obtained and found to be able to bind with rAP in ELISA assay.Then one of the 13 positive clone phage plasmid was used to infect E.coli HB2151 to induce the expression of the non-fusion single chain antibody (ScFv) with IPTG induction.As demonstrated by SDS-PAGE,the molecular mass of this single chain antibody was proved to be 30 kDa and the amount of expression constituted to 30% of the total bacterial proteins.Apparently,the human phage single chain antibody against surface protein of S.mutans with biological activity was successfully screened.

Objective To investigate the characteristics of intestinal microbiota in neonates on the first and third day after birth.Methods A total of 50 healthy singleton neonates who were born between June 15,2016 and August 3,2016 in Shanghai First Maternity and Infant Hospital were enrolled.Their stool samples were collected on the first and third day after birth and the samples were labeled according to the time of collection (D1 and D3 groups,n=50 each).Illumina NexSeq high-throughput sequencing platform was used to sequence the variable region 4 and 5 of all bacterial 16S rRNA genes in the samples.The composition of intestinal microbial communities was determined and the differences between the two groups were compared by Metastats analysis.Results (1) A total of 100 stool samples were sequenced and the retrieved sequences were from 25 bacterial phyla,119 families,227 genera and 159 species.(2) Major phyla in the two groups were the same,namely,Proteobacteria,Frimicutes,Bacteroidetes and Actinobacteria.The relative abundances of Frimicutes (0.27 ± 0.03 vs 0.41 ± 0.05) and Bacteroidetes (0.07 ± 0.01 vs 0.09 ± 0.03) increased over time,while that of Actinobacteria (0.10±0.01 vs 0.01 ±0.00) decreased on day 3.No significant difference in the relative abundance of Proteobacteria (0.51 ±0.03 vs 0.49± 0.05) was observed between D1 and D3 groups.There were significant difference in relative abundances of Frimicutes and Actinobacteria between the two groups (both q=-0.01,both P＜0.05).(3) Among the top ten most abundant families,Enterobacteriaceae,Staphylococcaceae,Enterococcaceae,Streptococcaceae and Lachnospiraceae were detected in both of the two groups.The relative abundances of Enterobacteriaceae (0.25 ± 0.02 vs 0.46 ± 0.06),Staphylococcaceae (0.07 ± 0.02 vs 0.12 ± 0.03),Enterococcaceae (0.04±0.02 vs 0.10±0.04),Streptococcaceae (0.03 ±0.02 vs 0.06±0.01) increased over time,while that of Lachnospiraceae (0.03 ± 0.01 vs 0.02 ± 0.02) decreased on day 3.Only the relative abundance of Enterobacteriaceae had statistical difference between the two groups (q=0.00,P＜0.05).(4) Among the top ten most abundant genera,Staphylococcus,Enterococcus,Streptococcus,Bacteroides and Pseudomonas were detected in both groups.The relative abundances of aerobic and facultative anaerobic bacteria which belonged to genera of Stenotrophomonas,Propionibacterium,Acinetobacter,Bacillus,Sphingomonas and so on decreased on day 3 as compared with those on day 1 (0.00±0.00 vs 0.07±0.02,0.00±0.00 vs 0.06±0.01,0.00±0.00 vs 0.03±0.01,0.00±0.00 vs 0.02±0.01,0.00±0.00 vs 0.02±0.00,all q=0.00,all P＜0.05).However,the relative abundances of anaerobic bacteria which belonged to Bacteroides,Veillonella,Parabacteroides and so on increased on day 3 (0.01 ±0.00 vs 0.08±0.03,0.00±0.00 vs 0.03±0.02,0.00±0.00 vs 0.01 ±0.00,q=0.01,0.01 and 0.00,all P＜0.05).(5) The most abundant species in intestinal microbiota was escherichia coli in both groups.Three less abundant species including lactobacillus gasseri,lactobacillus animalis and bifidobacterium bifidum were detected in both groups.(6) Regardless of the mode of delivery,Staphylococcus,was the highest predominant genera in meconium samples,followed by stenotrophomonas.Stool samples collected on the third day after birth were divided into four groups based on deliver modes and feeding patterns.Neonates who were born abdominally with exclusive breastfed thereafter were different from those of the other three groups in predominant intestinal bacteria,but the difference was not statistically significant.Bifidobacterium and Subdoligranulum were only detected in the vaginally born neonates.Conclusions Meconium is not sterile.Although the intestinal microbiota on the first day of life is different from that on the third day of life,the dominant bacteria are common.During the first three days of life,the relative abundances of aerobic and facultative anaerobic bacteria decreased significantly over time,while the relative abundance of anaerobic bacteria increased.

Humans ; Nails ; Hydroxyurea/adverse effects* ; Alopecia/chemically induced*

Objective: To investigate the status of passive smoking among pregnant women in Jinshan District, Shanghai Municipality, so as to provide insights into developing targeted smoking control measures and promoting maternal and infant health. Methods: Pregnant women who had early pregnancy registration at Jinshan District Community Health Service Center from April 2021 to December 2023 were selected as subjects. The basic information, passive smoking and awareness of passive smoking hazards among pregnant women were collected through questionnaire surveys, and passive smoking rate and awareness rate of passive smoking hazards were analyzed. Results: Totally 8 273 questionnaires were allocated, and 8 216 valid questionnaires were recovered, with an effective rate of 99.31%. The mean age of participants was (29.52±4.60) years. There were 4 991 participants with an education of college degree or above, accounting for 60.75%; 3 565 participants with the first pregnancy, accounting for 43.39%; 3 990 primiparas, accounting for 48.56%; 3 193 participants living with smokers, accounting for 38.86%. A total of 3 710 participants passively smoked, with a passive smoking rate of 45.16%. There were 2 817 participants passively smoked in public places, accounting for 75.93%; 2 253 participants passively smoked in workplaces, accounting for 60.73%; 1 563 participants that passively smoked at home, accounting for 42.13%. The awareness rates regarding the hazards of passive smoking to health, causing lung cancer in adults, causing lung diseases in children, causing preterm birth and low birth weight infants, and causing heart diseases in adults were 92.13%, 88.85%, 87.99%, 82.05% and 62.56%, respectively. Conclusion The rate of passive smoking among pregnant women in Jinshan District is comparatively high, while their awareness regarding non-respiratory diseases emanating from passive smoking is comparatively low.

The extract rates, multicomponent content and fingerprint were determined in this study to investigate the quality diffe-rence between standard decoction of raw Paeoniae Radix Alba and fried Paeoniae Radix Alba. UPLC fingerprint was established for 17 batches of standard decoction of raw and fried Paeoniae Radix Alba, and the contents of gallic acid, catechin, albiflorin, paeoniflorin and benzoyl paeoniflorin were determined. The peak areas of standard decoction were analyzed by the independent t-test and orthogonal partial least squares discriminant analysis. There was no significant difference in extract rates between the standard decoction of raw and fried Paeoniae Radix Alba. After fried processing, the content of albiflorin increased by 0.26%, while the contents of gallic acid, catechin, paeoniflorin and benzoyl paeoniflorin decreased by 13.04%, 27.97%, 10.30% and 18.79% respectively. There were 14 common peaks in the fingerprint of standard decoction of raw Paeoniae Radix Alba, and 16 common peaks in the fried Paeoniae Radix Alba. Peak 1 and peak 3 were new ones after processing, among which the peak 3 was 5-hydroxymethylfurfural. The results showed that peak 1, peak 3, peak 11 and peak 15 were the key compounds to distinguish standard decoction of raw and fried Paeoniae Radix Alba. In conclusion, this method is stable and can be used for the study of quantity transfer and quality control in the preparation process of standard decoction, granules and other dosage forms for raw and fried Paeoniae Radix Alba, providing reference for the identification of raw and fried Paeoniae Radix Alba and related preparations.
Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; Paeonia ; Quality Control ; Reference Standards

OBJECTIVE：To e stablish UPLC characteristics fingerprints of Lysimachia christinae ，and to simultaneously determine 3 effective components and to comprehensively evaluate the quality of L. christinae from different production areas. METHODS：UPLC method was adopted to establish characteristics fingerprint of the whole plant ，stem and leaves of 10 batches of L. christinae ，and determine the contents of kaemperfol- 3-O-rutinoside，quercetin，kaemperfol. The determination was performed on Waters CORTECS UPLC T 3 column with mobile phase consisted of acetonitrile- 0.2％ phosphoric acid aqueous solution （gradient elution ）at the flow rate of 0.2 mL/min. The detection wavelength was set at 364 nm，and column temperature was 30 ℃. The sample size was 1 µL. Similarity Evaluation System for TCM Chromatographic Fingerprint （2012 edition）was adopted to evaluate its similarity ， and common peaks were confirmed. Using the contents of kaemperfol- 3-O-rutinoside， quercetin， kaemperfol，total ash ，acid-insoluble ash and sulfur dioxide residue ，the ethanol-soluble extract as index ，entropy weight TOPSIS was used to evaluate the overall quality of L. christinae comprehensively. RESULTS ：There were 7 common peaks in the whole plant，stem and leaves of 10 batches of L. christinae ，among which 3 peaks were identified as kaemperfol- 3-O-rutinoside， quercetin and kaemperfol. The similarity of same part in the whole plant of L. christinae from different batches were not lower than 0.830. The similarity between stem and leaves of L. christinae in same batch was 0.504-0.859； the similarity between whole plant and stem was 0.593-0.904；the similarity between whole plant and leaves was 0.885-0.995. The linear ranges were 0.392 0-39.197 0 μg/mL for kaempferol-3-O-rutinoside， 0.397 0- 39.703 4 μg/mL for quercetin，0.380 9-38.093 0 μg/mL for kaempferol（r＞0.999 0）. RSDs of precision ，stability and repeatability tests were all lower than 2％. The recoveries were 96.43％（RSD＝0.63％，n＝9），100.32％（RSD＝0.46％，n＝9）， 101.80％（RSD＝0.32％，n＝9），respectively. The content range of above components in L. christinae were 0.006 3％-0.041 1％， 0.002 9％-0.008 6％，0.004 4％-0.017 5％（stem）；0.024 8％-0.290 5％，0.000 9％-0.009 0％，0.001 3％-0.012 4％（leaves）； 0.007 9％-0.118 0％，0.001 5％-0.008 8％，0.002 8％-0.012 5％（whole plant ）. There was no significant difference in the contents of 3 components in L. christinae among different producing areas （P＞0.05）. The order of the contents of kaempferol- 3-O- rutinoside in different parts of L. christinae was leaves ＞whole plant ＞stem. The contents of quercetin and kaempferol were high relatively in the stem. Results of entropy weight TOPSIS method showed that mean values of Ci for L. christinae from Zhongjiang county and Shuangliu county of Sichuan province ，Shizhu county of Chongqing city were 0.446，0.512，0.287. CONCLUSIONS ： Established fingerprint and content determination method are stable and feasible ，and multi-index evaluation model constructed by characteristic chromatogram combined with entropy weight TOPSIS analysis method can be used for comprehensive quality evaluation of L. christinae . The quality of L. christinae from Sichuan province is better.

Methods:Cultured human alveolar epithelial A549 cells were assigned into LPS group and blank control group. LPS group was stimulated with LPS and adenosine triphosphate to induce pyroptosis and inflammation. A549 cells were divided into 4 groups: miR-20a mimics group, mimics-negative control (NC) group, inhibitor group and inhibitor-NC group. MiRNA-20a mimics, mimics-NC, inhibitor, and inhibitor-NC were transfected respectively into A549 cells, and after 24 h, the cells were collected to verify transfection efficiency by qPCR. MiRNA-20a mimics and the constructed TLR4-3'UTR double luciferase reporter plasmid were co-transfected into A549 cells, and luciferase activity was analyzed. MiRNA-20a mimics/inhibitors were transfected into A549 cells, and then the cells were stimulated by LPS for 8 h followed by adenosine triphosphate for 30 min. QPCR, Western Blot and ELISA were used to detect the expression of GSDMD, inflammatory factors (ASC, NLRP3, Caspase-1, IL-1β) and Signaling molecules (TLR4、NF-κB) in A549 cells at mRNA level and protein level. Immunofluorescence was used to detect the expression of TLR4 in the A549 cells and NF-κB in the nucleus of A549 cells after transfecting with miRNA-20a mimics/inhibitor.Results:The mRNA and protein expression of pyroptosis marker molecule (GSDMD) and inflammatory factors (ASC, NLRP3, Caspase-1, IL-1β) in A549 cells stimulated with LPS were significantly higher than those in the blank control group, and the differences were statistically significant ( P<0.05). The expression of miRNA-20 in the mimics group was significantly higher than that in the mimic-NC group ( P<0.05), while the expression of miRNA-20a in the inhibitor group was lower than that in the inhibitor-NC group ( P<0.01). The double luciferase reporter gene experiment showed that the relative fluorescence value of the co-transfection group for TLR4-3'UTR-WT and miRNA-20a mimics was significantly lower than the co-transfection group for TLR4-3'UTR-WT and miRNA-20a mimics-NC ( P<0.05). The mRNA and protein levels of pyroptosis marker molecule (GSDMD) , inflammatory factors (ASC, NLRP3, Caspase-1, IL-1β) and signaling molecules (TLR4, NF-κB) were decreased in the mimics group compared to the mimics-NC group, and increased in inhibitor group compared to inhibitor-NC group. Conclusions:miRNA-20a may inhibit LPS-induced pyroptosis and inflammation of A549 cells via TLR4/NF-κB signal pathway.Objetive:To explore the potential role of miRNA-20a in lipopolysaccharide (LPS) induced pyroptosis and inflamation of human alveolar epithelial A549 cells and its regulation mechanisim.

The establishment and development of the intestinal microbiota in early life is critical to the development of intestinal immune system, digestive function, metabolic function, and central nervous system function in infants.The first 1000 days of life is a critical period for the establishment of intestinal microbiota, which is susceptible to many factors.The disorder of intestinal microbiota in this period will increase the risk of some diseases in the long-term life.In this review, we will describe the main physiological functions of the early-life microbiota and its role in programming future health of infants.We also will describe the establishment and development of the intestinal microbiota in early life and its main drivers.

Objective:To evaluate the clinical effect of autologous nanofat combined with pearl fat transplantation in comprehensive improvement of lacrimal groove depression.Methods:Seventy-eight patients (age ranges from 28 to 56 years, with average 38 years) who desired for lacrimal groove improvement were involved in this study from Jan. 2019 to Jun. 2020 in the Department of Plastic Surgery in Peking Union Medical College Hospital. Primary fat tissue was obtained and purified by liposuction. Nanofat and pearl fat were prepared and injected into lacrimal groove area in different layers and multiple points evenly to ameliorate depression. Visual analogue scoring (VAS) was used for evaluating injection pain. Dark eye circles, faint lines on lower eyelid, color spots and lacrimal groove depression between pre-operation pictures and post-operation pictures of 1 year follow-up were evaluated by patients＇ satisfaction scores.Results:All 78 patients revealed mild swelling and disappeared in 3 to 4 days. Injection areas were stable in 3 months. All patients appeared no complications such as infection, hematoma, fat liquefaction, local induration and so on. Among 78 patients, 2 patients showed ecchymosis after surgery which disappeared in 10 days, and 1 patient showed uneven appearance which disappeared after timely treatment. After 1 year of follow-up, the average satisfaction score of improvement was 8.9±0.5, which showed satisfied post-operative effect.Conclusions:Autologous nanofat combined with pearl fat transplantation has high feasibility, short operation time, which could achieve good effect of facial rejuvenation with high patients＇ satisfaction. In this case, this technique is worthy of clinical promotion.