Objective:To discuss the effect of quorum sensing-related gene expression on biofilm formation and drug resistance in clinically multidrug-resistant Pseudomonas aeruginosa,and to clarify the mechanism of enhacing drug resistance.Methods:A total of 77 strains of Pseudomonas aeruginosa were collected.Based on drug resistance,the strains were divided into multidrug-resistant group and sensitive group.The optimal biofilm formation conditions were determined using the microtiter plate method;biofilm formations of the stains in both groups was observed under an optical microscope;crystal violet staining was used to semiquantitatively detect biofilm formation ability of P.aeruginosa in both groups;microbroth dilution method was used to determine the minimal inhibitory concentration(MIC)values of the quorum sensing inhibitor(C-30)against Pseudomonas aeruginosa in both groups;RNA was extracted from two groups using a commercial kit,while RNA from planktonic state and biofilm state of multidrug-resistant strains was extracted using modified TRIzol method;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the mRNA expression levels of quorum sensing-related genes(lasR/I,RhlR/I,PqsR/A)of the stains in multidrug-resistant group and sensitive group,as well as before and after adding the quorum sensing inhibitor C-30.Results:Among 77 strains of Pseudomonas aeruginosa,56 were multidrug-resistant(multidrug-resistant group)and 21 were fully sensitive(sensitive group).Optimal biofilm formation occurred at a bacterial concentration of 1.5×108 CFU·mL-1 with 48 h incubation.The biofilm positivity rate was 91%,with strongly positive,moderately positive,weakly positive,and negative biofilms accounting for 16%,34%,41%,and 9%,respectively.The biofilm positivity rate in multidrug-resistant strains was 96%,and biofilm formation ability in multidrug-resistant group was higher than that in sensitive group(P<0.05).When the concentration of C-30 was 8 mg·L-1 the biofilm formation in most Pseudomonas aeruginosa was inhibited,with enhanced suppression at higher concentrations.The absorbtion(A)value of both planktonic-state and biofilm-state RNA ranged from 1.8 to 2.0.The RT-qPCR results showed that compared with planktonic state,the expression levels of lasR/I,RhlR/I,and PqsR/A mRNA of the stains in biofilm state were significantly increased(P<0.01).Compared with non-inhibitor group,the expression levels of lasR/I,RhlR/I,and PqsR/A mRNA in biofilm state of inhibitor-treated group were significantly decreased(P<0.01).Conclusion:High expression of quorum sensing-related genes in multidrug-resistant P.aeruginosa promotes biofilm formation,thereby enhancing drug resistance.

Objective:To establish a fluorescent quantitative RT-PCR assay based on N_sgRNA of SARS-CoV-2 and preliminarily apply it on real samples.Methods:Recombinant plasmid, specific primers and probes of N_sgRNA were designed and synthesized based on Wuhan-Hu-1/2019_MN908947 and synthesis mechanism of subgenomic RNA (sgRNA). Using recombinant plasmid as amplification templates, the optimal reaction conditions and reaction system were screened according to the Ct value, fluorescence intensity, and shape of amplification curve and was evaluated for sensitivity, reproducibility, and specificity. Meanwhile, the possibility of practical application of the method was explored by testing 172 clinical samples and 256 municipal wastewater samples. Results:A qRT-PCR assay for N_sgRNA in SARS-CoV-2 was initially established. The detection limit of the assay was 20 copies/mL, and the variation coefficients of in-batch (0.002%~0.767%) and batch to batch repetition (0.016%~0.752%) were less than 1%. Only N_sgRNA recombinant plasmid was detected in the specificity assay. So the method is more highly sensitive, specific and reproducible. The RatiosgRNA/ gRNA of clinical samples HK.3, EG.5.1, JN.1 and their sub-lineages and their corresponding urban sewage samples in epidemic period were significantly different ( P<0.05). There is a strong correlation between the median of RatiosgRNA/ gRNA in clinical samples and sewage samples in the same period (correlation coefficient r=1.000, P=0.010). Conclusions:In this study, a qRT-PCR method for detecting N_sgRNA of SARS-CoV-2 was established and the method has the characteristics of higher sensitivity, stronger specificity and better repeatability, and it can be used to detect SARS-CoV-2 infectivity.

Objective:To establish a fluorescent quantitative RT-PCR assay based on N_sgRNA of SARS-CoV-2 and preliminarily apply it on real samples.Methods:Recombinant plasmid, specific primers and probes of N_sgRNA were designed and synthesized based on Wuhan-Hu-1/2019_MN908947 and synthesis mechanism of subgenomic RNA (sgRNA). Using recombinant plasmid as amplification templates, the optimal reaction conditions and reaction system were screened according to the Ct value, fluorescence intensity, and shape of amplification curve and was evaluated for sensitivity, reproducibility, and specificity. Meanwhile, the possibility of practical application of the method was explored by testing 172 clinical samples and 256 municipal wastewater samples. Results:A qRT-PCR assay for N_sgRNA in SARS-CoV-2 was initially established. The detection limit of the assay was 20 copies/mL, and the variation coefficients of in-batch (0.002%~0.767%) and batch to batch repetition (0.016%~0.752%) were less than 1%. Only N_sgRNA recombinant plasmid was detected in the specificity assay. So the method is more highly sensitive, specific and reproducible. The RatiosgRNA/ gRNA of clinical samples HK.3, EG.5.1, JN.1 and their sub-lineages and their corresponding urban sewage samples in epidemic period were significantly different ( P<0.05). There is a strong correlation between the median of RatiosgRNA/ gRNA in clinical samples and sewage samples in the same period (correlation coefficient r=1.000, P=0.010). Conclusions:In this study, a qRT-PCR method for detecting N_sgRNA of SARS-CoV-2 was established and the method has the characteristics of higher sensitivity, stronger specificity and better repeatability, and it can be used to detect SARS-CoV-2 infectivity.

ObjectiveTo investigate the effect of Chaihu Guizhitang on triple-negative breast cancer （TNBC） cells based on hypoxia-inducible factor-1α （HIF-1α）/vascular endothelial growth factor A （VEGFA） signaling pathway. MethodTNBC xenograft model was established and the cells were randomized into model group， capecitabine group （0.2 mg·kg^-1）， Chaihu Guizhitang low-dose group， medium-dose group， and high-dose group （10.62， 21.23， 42.46 g·kg^-1）， with 10 mice in each group. After 21 days of medication， the content of tumor necrosis factor-α （TNF-α） in serum was detected by enzyme-linked immunosorbent assay （ELISA）. The expression of HIF-1α mRNA was detected by real-time fluorogenic quantitative polymerase chain reaction （real-time PCR）. Immunohistochemistry （IHC） was employed to detect the expression of HIF-1α， TNF-α， and VEGFA in tumor tissues， and CD34 staining to examine the angiogenesis in tumor tissues. Microvessel density （MVD） was calculated， and the protein expression of HIF-1α， VEGFA， and epidermal growth factor receptor （EGFR） in tumor tissues was measured by Western blot. ResultCompared with the model group， the rest four groups showed low levels of TNF-α （P<0.01）， HIF-1α mRNA （P<0.01）， expression of HIF-1α， TNF-α， VEGFA， and CD34 in cells， and MVD （P<0.05， P<0.01）， and low protein levels of HIF-1α， VEGFA， and EGFR （P<0.01）. Compared with capecitabine group， medium-dose and high-dose Chaihu Guizhitang decreased the level of TNF-α （P<0.01）， HIF-1α mRNA （P<0.01）， expression of HIF-1α， TNF-α， and VEGFA in cells （P<0.01）， CD34 expression， MVD， and protein levels of HIF-1α， VEGFA， and EGFR （P<0.01）. ConclusionChaihu Guizhitang may inhibit the angiogenesis in TNBC cells by regulating the expression of HIF-1α/VEGFA signaling pathway， thus exerting anti-tumor effect.

Objective:To study the genotyping of thalassemia in Ningbo population and provide a reference basis for future prevention and control of thalassemia in Ningbo.Methods:Patients with suspected thalassemia attending Ningbo Women and Children's Hospital from January 2019 to March 2022 were selected for the study, and DNA was extracted from dried blood spot specimens by collecting peripheral blood, and detection of thalassemia hotspot variants was performed by fluorescence PCR melting curve analysis combined with Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA).Results:A total of 2 680 cases were included in the patients with suspected thalassemia, and 1 426 cases of thalassemia gene carriers were detected, with an overall detection rate of 53.2%. Among them, 595 cases (41.7%) were α-thalassemia, with -- SEA/αα, αα/-α 3.7 and -- SEA/-α 3.7 being more common; 807 cases (56.6%) were β-thalassemia, with β IVS-Ⅱ-654/β N, β CD41-42/β N and β CD17/β N being more common; 24 cases (1.7%) were αβ-combined thalassemia. Among them, six rare variant types were included, including fusion gene (Fusion), -- FIL, HBA2:c.376C>T, CD8/9(+G), IVS-Ⅰ-2(T>C) and IVS-Ⅱ-1(G>A), all of which were reported for the first time in Ningbo. Conclusion:Among suspected thalassemia patients in Ningbo, the detection rate of thalassemia is high, and the types of gene variants are complex, so the awareness of thalassemia gene testing for anemic patients should be raised.

Objective:To identify the transmission relationship between HIV infection cases the non-marital non-commercial heterosexual contact in Zhejiang Province.Methods:When HIV positive was informed during January 2020 to January 2022, the staff conducted an epidemiological investigation to collect cases information on sociodemographic characteristics, mobility information, past HIV testing history, high-risk sexual behaviors, sexual partners, and etcetera. At the same time, 6-8 ml of blood from the new diagnosis of people infected with HIV before antiviral treatment was collected to separate the bleeding plasma. pol gene was amplified by nucleic acid extraction and PCR, sequenced by Sequencer 5.0 software, and Cytoscape 3.6.0 software was used to draw HIV molecular transmission network. Results:From January 2020 to January 2022, 88 HIV infected individuals were found in Pujiang County, of which 74 were transmitted through heterosexual transmission, of which 31 were infected through non-marital non-commercial heterosexual contact. Preliminary case studies have found that three female cases have engaged in unprotected non-marital non-commercial heterosexual contact with one male case. Among the 4 infected individuals, 2 of their spouses tested positive for HIV antibodies. Molecular transmission network monitoring was carried out on 65 newly diagnosed cases of heterosexual transmission with acquired sequences, forming 9 transmission clusters. The largest cluster contained 10 cases. A total of 11 HIV-infected individuals were involved in this HIV cluster epidemic. They were 3 males and 8 females, all over 50 years old and were farmers or rural housewives. They were traced to 7 sexual partners (6 negatives of HIV, 1 undetected). Among the 18 respondents' sexual social network relationships, there were 6 couples, 8 permanent partners, and 3 temporary partners. Among 11 HIV infected individuals, there were 9 cases of non-marital non-commercial heterosexual transmission and 2 cases of intramarital transmission. The epidemiological association between 7 non-married non-commercial heterosexual partners and case 2 (56-year-old male farmer), 3 cases confirmed by epidemiological investigation and molecular transmission cluster results, 3 cases confirmed by molecular transmission cluster and epidemiological investigation results, and 1 case confirmed by epidemiological investigation results.Conclusions:The transmission mode of this cluster epidemic was to spread HIV through heterosexual sex with a male case as the core, then cause the transmission within marriage and between fixed sexual partners. The combination of epidemiological investigation and molecular transmission network traceability survey supports the conclusion of this study.

ObjectiveTo investigate the current situation of personality traits and job satisfaction of pediatric therapists in Jiangsu province, and to explore the correlation between them and the related factors of job satisfaction. MethodsFrom June, 2021 to February, 2022, random cluster sampling method was adopted after field investigating several pediatric hospitals and institutions in Jiangsu. Online questionnaires were sent out to pediatric therapists in different settings in eleven cities in Jiangsu. The questionnaires included Minnesota Satisfaction Questionnaire (MSQ), Eysenck Personality Questionnaire-Revised Short Scale for Chinese (EPQ-RSC) and self-made scale of demographic characteristics and working status. ResultsAll of the questionaires were taken back, in which 165 were valid (83.33%). Among the three dimensions of psychoticism, extraversion and neuroticism, the intermediate type accounted for 66.06%, 38.79% and 40.00%, respectively. The overall satisfaction of job was (3.80±0.55), the internal satisfaction was (3.88±0.53), and the external satisfaction was (3.59±0.69). There was significant difference in the satisfaction among different types of work institution, monthly incomes, levels of parental support and neuroticism (P < 0.05). Multiple linear regression showed that the internal satisfaction and overall satisfaction were related with neuroticism, monthly income and parental support (P < 0.05), while external satisfaction was related with the neuroticism and parental support (P < 0.05). The level of neuroticism was negatively correlated with the internal satisfaction, the external satisfaction and overall satisfaction (|r| > 0.337, P < 0.001), and the level of extraversion was postively correlated with the external satisfaction (r = 0.155, P < 0.05). ConclusionThe job satisfaction of pediatric therapists is fine in Jiangsu, and is ralated with the level of neuroticism, the monthly income and the level of parental support. The levels of neuroticism and extraversion correlate with the job satisfaction.

OBJECTIVE：To systematically evaluate the research and development status quo of respiratory inhalation preparations. METHODS ：Related literatures or data about R&D status quo of domestic and foreign respiratory inhalation preparations were retrieved from 5 databases（PubMed，Embase，CNKI，Wanfang database and VIP ），4 clinical trial registration platforms，15 national/regional drug administration platforms and yaozhi.com . The status quo of related studies was analyzed by descriptive analysis. RESULTS & CONCLUSIONS ：A total of 27 second literatures were included. The information of respiratory inhalation preparations approved by European Union ，the United States ，Canada，Japan and China was collected ，and the information of related drugs under registration and approval in China was also collected. The market situation of respiratory inhalation preparations was analyzed from the perspective of dosage form ：powder aerosols accounted for 46％，aerosols account ed for 32％，and atomized inhalation solutions accounted for 22％ in the global consumption sum of each dosage form. From the perspective of market share ，companies such as AstraZeneca in the UK ，Boehringer Ingelheim in Germany and GlaxoSmithKline in the UK had a total market share of about 70％. At present ，there were 39 kinds of respiratory inhalation preparations on the market in European Union ，121 kinds in the United States ，111 kinds in Canada ，37 kinds in Japan ，69 kinds of domestic inhalation preparations and 80 kinds of imported inhalation preparations in China. Respiratory inhalation preparations were undergoing clinical trials with 511 cases abroad and 69 cases in China. The barriers to the imitation of respiratory inhalation preparations mainly included research and development ，clinical trial ，approval and production barriers. At present ，the support is provided for 0035） domestic drug innovation and localization of medical devices in China through a series of policies ，which is helpful to promote the localization research and development of inha led preparations. Future research and development can pay more cn attention to combination ，indication，dosage and device of inhalation preparation.

Background: Our previously prepared ceftiofur (CEF) hydrochloride oily suspension shows potential wide applications for controlling swine Streptococcus suis infections, while the irrational dose has not been formulated. Objectives: The rational dose regimens of CEF oily suspension against S. suis were systematically studied using a pharmacokinetic-pharmacodynamic model method. Methods: The healthy and infected pigs were intramuscularly administered CEF hydrochloride oily suspension at a single dose of 5 mg/kg, and then the plasma and pulmonary epithelial lining fluid (PELF) were collected at different times. The minimum inhibitory concentration (MIC), minimal bactericidal concentration, mutant prevention concentration (MPC), post-antibiotic effect (PAE), and time-killing curves were determined. Subsequently, the area under the curve by the MIC (AUC 0–24h /MIC) values of desfuroylceftiofur (DFC) in the PELF was obtained by integrating in vivo pharmacokinetic data of the infected pigs and ex vivo pharmacodynamic data using the sigmoid E max (Hill) equation. The dose was calculated based on the AUC 0–24h /MIC values for bacteriostatic action, bactericidal action, and bacterial elimination. Results: The peak concentration, the area under the concentration-time curve, and the time to peak for PELF's DFC were 24.76 ± 0.92 µg/mL, 811.99 ± 54.70 μg·h/mL, and 8.00 h in healthy pigs, and 33.04 ± 0.99 µg/mL, 735.85 ± 26.20 μg·h/mL, and 8.00 h in infected pigs, respectively. The MIC of PELF's DFC against S. suis strain was 0.25 µg/mL. There was strong concentration-dependent activity as determined by MPC, PAE, and the time-killing curves. The AUC 0–24h /MIC values of PELF's DFC for bacteriostatic activity, bactericidal activity, and virtual eradication of bacteria were 6.54 h, 9.69 h, and 11.49 h, respectively. Thus, a dosage regimen of 1.94 mg/kg every 72 h could be sufficient to reach bactericidal activity. Conclusions A rational dosage regimen was recommended, and it could assist in increasing the treatment effectiveness of CEF hydrochloride oily suspension against S. suis infections.

OBJECTIVE: To evaluate the secretory function of parotid glands by dynamic magnetic resonance (MR) sialography and determine the clinical performance of this technique in diagnosing and evaluating Sjögren's syndrome (SS) patients. MATERIALS AND METHODS: This study enrolled 29 healthy volunteers (25 women and 4 men; mean age, 34.8 ± 6.3 years; age range, 26–47 years) and 25 primary SS (pSS) patients (23 women and 2 men; mean age, 37.7 ± 7.9 years; age range, 25–50 years) with decreased secretory function. The volume of the parotid gland ducts was precisely measured for both groups at single pre- and 6 post-gustatory-stimulated phases. Time-dependent volume change ratio curves were generated, four parameters were derived from the curves: the slope of the increase in the first post-stimulation phase (slope(1st)), the peak value, the time-to-peak, the total saliva secretion post-stimulation. All values were used to quantitatively evaluate the secretory function of the parotid gland. The repeated measurement analysis, Mann-Whitney U test and receiver operating characteristic curve were applied. RESULTS: Time-dependent volume change ratio curves demonstrated that there is a statistically significant difference between the two groups (F = 8.750; p = 0.005). A quickly increasing curve was shown in the volunteer group, whereas a slowly increasing curve was shown in the pSS patient group. The slope(1st), peak value and total saliva secretion post-stimulation of the patient group were significantly lower than those of the volunteer group (p = 0.005, p = 0.003, and p = 0.002, respectively). The time-to-peak between the two groups was not significantly different (p = 0.383). The slope(1st) can be used as a discriminator to diagnose SS patients (p = 0.015; odds ratio = 4.234; area under the curve = 0.726). CONCLUSION: Dynamic MR sialography is proven to be an effective method in evaluating salivary gland function and has a great potential in diagnosing and evaluating pSS patients.
Autoimmune Diseases ; Female ; Healthy Volunteers ; Humans ; Male ; Methods ; Odds Ratio ; Parotid Gland ; ROC Curve ; Saliva ; Salivary Glands ; Salivation ; Sialadenitis ; Sialography ; Volunteers