OBJECTIVES: To explore the optimal maintenance dose of caffeine citrate for preterm infants requiring assisted ventilation and caffeine citrate treatment. METHODS: A retrospective analysis was performed on the medical data of 566 preterm infants (gestational age ≤34 weeks) who were treated and required assisted ventilation and caffeine citrate treatment in the neonatal intensive care unit of 30 tertiary hospitals in Jiangsu Province of China between January 1 and December 31, 2019. The 405 preterm infants receiving high-dose (10 mg/kg per day) caffeine citrate after a loading dose of 20 mg/kg within 24 hours after birth were enrolled as the high-dose group. The 161 preterm infants receiving low-dose (5 mg/kg per day) caffeine citrate were enrolled as the low-dose group. RESULTS: Compared with the low-dose group, the high-dose group had significant reductions in the need for high-concentration oxygen during assisted ventilation (P=0.044), the duration of oxygen inhalation after weaning from noninvasive ventilation (P<0.01), total oxygen inhalation time during hospitalization (P<0.01), the proportion of preterm infants requiring noninvasive ventilation again (P<0.01), the rate of use of pulmonary surfactant and budesonide (P<0.05), and the incidence rates of apnea and bronchopulmonary dysplasia (P<0.01), but the high-dose group had a significantly increased incidence rate of feeding intolerance (P=0.032). There were no significant differences between the two groups in the body weight change, the incidence rates of retinopathy of prematurity, intraventricular hemorrhage or necrotizing enterocolitis, the mortality rate, and the duration of caffeine use (P>0.05). CONCLUSIONS This pilot multicenter study shows that the high maintenance dose (10 mg/kg per day) is generally beneficial to preterm infants in China and does not increase the incidence rate of common adverse reactions. For the risk of feeding intolerance, further research is needed to eliminate the interference of confounding factors as far as possible.
Caffeine/therapeutic use* ; Citrates ; Humans ; Infant ; Infant, Newborn ; Infant, Premature ; Respiration, Artificial ; Retrospective Studies

Objective: To compare the prognostic evaluation value of preoperative neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), lymphocyte-to-monocyte ratio (LMR), and systemic immune-inflammation index (SII) in rectal cancer patients. Nomogram survival prediction model based on inflammatory markers was constructed. Methods: The clinical and survival data of 585 patients with rectal cancer who underwent radical resection in the First Affiliated Hospital of Xi'an Jiao tong University from January 2013 to December 2016 were retrospectively analyzed. The optimal cut-off values of NLR, PLR, LMR, and SII were determined by the receiver operating characteristic (ROC) curve. The relationship between different NLR, PLR, LMR and SII levels and the clinic pathological characteristics of the rectal cancer patients were compared. Cox proportional risk model was used for univariate and multivariate regression analysis. Nomogram prediction models of overall survival (OS) and disease-free survival (DFS) of patients with rectal cancer were established by the R Language software. The internal validation and accuracy of the nomograms were determined by the calculation of concordance index (C-index). Calibration curve was used to evaluate nomograms' efficiency. Results: The optimal cut-off values of preoperative NLR, PLR, LMR and SII of OS for rectal cancer patients were 2.44, 134.88, 4.70 and 354.18, respectively. There was statistically significant difference in tumor differentiation degree between the low NLR group and the high NLR group (P<0.05), and there were statistically significant differences in T stage, N stage, TNM stage, tumor differentiation degree and preoperative carcinoembryonic antigen (CEA) level between the low PLR group and the high PLR group (P<0.05). There was statistically significant difference in tumor differentiation degree between the low LMR group and the high LMR group (P<0.05), and there were statistically significant differences in T stage, N stage, TNM stage, tumor differentiation degree and preoperative CEA level between the low SII group and the high SII group (P<0.05). The multivariate Cox regression analysis showed that the age (HR=2.221, 95%CI: 1.526-3.231), TNM stage (Ⅲ grade: HR=4.425, 95%CI: 1.848-10.596), grade of differentiation (HR=1.630, 95%CI: 1.074-2.474), SII level (HR=2.949, 95%CI: 1.799-4.835), and postoperative chemoradiotherapy (HR=2.123, 95%CI: 1.506-2.992) were independent risk factors for the OS of patients with rectal cancer. The age (HR=2.107, 95%CI: 1.535-2.893), TNM stage (Ⅲ grade, HR=2.850, 95%CI: 1.430-5.680), grade of differentiation (HR=1.681, 95%CI: 1.150-2.457), SII level (HR=2.309, 95%CI: 1.546-3.447), and postoperative chemoradiotherapy (HR=1.837, 95%CI: 1.369-2.464) were independent risk factors of the DFS of patients with rectal cancer. According to the OS and DFS nomograms predict models of rectal cancer patients established by multivariate COX regression analysis, the C-index were 0.786 and 0.746, respectively. The calibration curve of the nomograms showed high consistence of predict and actual curves. Conclusions: Preoperative NLR, PLR, LMR and SII levels are all correlated with the prognosis of rectal cancer patients, and the SII level is an independent prognostic risk factor for patients with rectal cancer. Preoperative SII level can complement with the age, TNM stage, differentiation degree and postoperative adjuvant chemoradiotherapy to accurately predict the prognosis of rectal cancer patients, which can provide reference and help for clinical decision.
Biomarkers, Tumor ; Carcinoembryonic Antigen ; Humans ; Inflammation/classification* ; Lymphocytes ; Neutrophils ; Nomograms ; Preoperative Period ; Prognosis ; Rectal Neoplasms/surgery* ; Retrospective Studies

Objective:To seed for stable time window of the integrated disease-syndrome animal model based on the counterevidence from Chinese medicinal prescriptions， and to verify syndrome stability and reliability. Method:A model of depression was established by exposing rats to chronic unpredictable mild stress （CUMS）， followed by body weight measurement， sugar water test， behavioral test， and brain 5-hydroxytryptamine（5-HT） detection. The identification of liver depression and spleen deficiency syndrome was conducted after the equivalent transformation of human clinical symptoms into macroscopic representations of rats. Based on the dynamically collected macroscopic representation scale， Xiaoyaosan was used to reversely verify the stability and reliability of the integrated disease-syndrome animal model of depression due to liver depression and spleen deficiency. Result:The sugar water consumption and the number of crossings and the total movement distance in the open field test of 16-week-old rats in the CUMS （eight weeks of CUMS） group were significantly lower than those in the normal group （P<0.05）. According to the immunohistochemical results， the 5-HT content in hippocampal area CA2 of rats in the CUMS group was also significantly lowered as compared with that in the normal group（P<0.05），which indicated that depression was successfully modeled. The liver depression and spleen deficiency syndrome was present in 14-week-old rats （six weeks after CUMS）of the CUMS group， and the number of rats experiencing the liver depression and spleen deficiency syndrome reached the peak in the 16th week （eight weeks after CUMS），accounting for 70% of the total number. Thereafter， the number decreased gradually. The syndrome scores of the 14-， 16-， 18-， 20-， and 22-week-old rats in the Xiaoyaosan group were reduced by 66.6%， 70.7%， 54.8%， 50.4%， and 44.8%， which were graded as effective， marked effective， effective， effective， and effective， respectively. Conclusion:The age of 14-16 weeks（six to eight weeks after CUMS） is considered the stable and reliable time window for depression due to liver depression and spleen deficiency.

BACKGROUND: Circulating tumor DNA (ctDNA) is a promising biomarker for non-invasive epidermal growth factor receptor mutations (EGFRm) detection in lung cancer patients, but existing methods have limitations in sensitivity and availability. In this study, we used the ΔCt value (mutant cycle threshold [Ct] value-internal control Ct value) generated during the polymerase chain reaction (PCR) assay to convert super-amplification-refractory mutation system (superARMS) from a qualitative method to a semi-quantitative method named reformed-superARMS (R-superARMS), and evaluated its performance in detecting EGFRm in plasma ctDNA in patients with advanced lung adenocarcinoma. METHODS: A total of 41 pairs of tissues and plasma samples were obtained from lung adenocarcinoma patients who had known EGFRm in tumor tissue and were previously untreated. EGFRm in ctDNA was identified by using superARMS. Through making use of ΔCt value generated during the detection process of superARMS, we indirectly transform this qualitative detection method into a semi-quantitative PCR detection method, named R-superARMS. Both qualitative and quantitative analyses of the data were performed. Kaplan-Meier analysis was performed to estimate the progression-free survival (PFS) and overall survival (OS). Fisher exact test was used for categorical variables. RESULTS: The concordance rate of EGFRm in tumor tissues and matched plasma samples was 68.3% (28/41). At baseline, EGFRm-positive patients were divided into two groups according to the cut-off ΔCt value of EGFRm set at 8.11. A significant difference in the median OS (mOS) between the two groups was observed (EGFRm ΔCt ≤8.11 vs. >8.11: not reached vs. 11.0 months; log-rank P = 0.024). Patients were divided into mutation clearance (MC) group and mutation incomplete clearance (MIC) group according to whether the ΔCt value of EGFRm test turned negative after 1 month of treatment. We found that there was also a significant difference in mOS (not reached vs. 10.4 months; log-rank P = 0.021) between MC group and MIC group. Although there was no significant difference in PFS between the two groups, the two curves were separated and the PFS of MC group tended to be higher than the MIC group (not reached vs. 27.5 months; log-rank P = 0.088). Furthermore, EGFRm-positive patients were divided into two groups according to the cut-off of the changes in ΔCt value of EGFRm after 1 month of treatment, which was set at 4.89. A significant difference in the mOS between the two groups was observed (change value of ΔCt >4.89 vs. ≤4.89: not reached vs. 11.0 months; log-rank P = 0.014). CONCLUSIONS Detecting EGFRm in ctDNA using R-superARMS can identify patients who are more likely sensitive to targeted therapy, reflect the molecular load of patients, and predict the therapeutic efficacy and clinical outcomes of patients.
Adenocarcinoma of Lung/genetics* ; Circulating Tumor DNA/genetics* ; ErbB Receptors/genetics* ; Humans ; Lung Neoplasms/genetics* ; Mutation/genetics* ; Protein Kinase Inhibitors

In order to reveal the molecular mechanism of blue labeled genic male sterility (BM-type GMS) and utilize the heterosis of BM-type GMS, we used the anthers of white-seed plants WS (sterile) and light blue seed plants WF (normal fertility) as experimental materials to analyze the differences in gene expression between them by transcriptome technology. And we also verified the genes expressed in anthocyanin synthesis in this study. Compared with WF, a total of 2352 differentially expressed genes were detected in WS. According to GO functional annotation, these genes could be divided into 3 categories and 43 subgroups. They are mainly involved in biosynthesis, phenylpropane metabolism, L-phenylalanine catabolism, membrane components, plasma membrane, cytoplasm, ATP binding, protein serine/threonine kinase activity, etc. KEGG pathway analysis showed that there were 159 genes enriched in the phenylpropanoid biosynthesis pathway, followed by the phenylalanine pathway, including 136 differentially expressed genes. Other genes are also involved a variety of amino acid metabolism, purine metabolism, pyrimidine metabolism and sugar metabolism pathway. Related to anthocyanin metabolism, several structural genes of key enzymes were differentially expressed, and most of them were up-regulated in WF, while only Flavanone 3-hydroxylase (F3H) and colorless anthocyanin dioxygenase (ANS) were down-regulated. Quantitative real-time PCR showed that the expression of 10 genes related to anthocyanin metabolism had the same trend as that in transcriptome sequencing data. Sequence homology analysis showed that the two selected transcription factors (DN48762c2g1 and DN25944c0g1) are clustered into the same cluster as the transcription factors regulating anthocyanin biosynthesis in maize, rice and Arabidopsis thaliana, which might be candidate genes for the blue aleurone layer of light blue seed plants in wheat. And fluorescence quantitative analysis showed that the expression level of DN48762c2g1 and DN25944c0g1 in WF was significantly higher than that in WS. In conclusion, the genes related to the anthocyanin biosynthesis pathway are not only related to the blue grain trait, but also may be involved in the anther abortion of BM-type GMS.

Epithelial cell adhesion molecule (EpCAM) is a popular target for cancer therapy. In this research, 3 nanobodies with high specificity and endocytosis activity against EpCAM were developed, which provides a basis for the study of immunotoxin based on EpCAM. In our preliminary experiments, we have immunized a camel with EpCAM-Fc antigen and constructed a high-quality phage display library. Seventeen nanobodies with different complementarity determining region (CDR) 3 sequences have been screened after 3 rounds of biopanning by phage display technology. The animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Fudan University School of Pharmacy. After purification, 7 nanobodies showed high cell binding activity by fluorescent activated cell sorting (FACS) identification. Furthermore, 3 nanobodies presented high endocytosis activity based on FACS and laser confocal microscopy, which also showed high affinity to EpCAM measured by ForteBio. According to this study, we aimed to provide a novel alternative approach to the EpCAM-targeted therapy and to provide guidance for the study of nanobody based immunotoxins for other targets.

Objective:To observe the effect of combination of Tanreqing injection(Tanreqing) and imipenem-cilastatin on extensively-drug resistant Pseudomonas aeruginosa (XDPA), and study the mechanism of the combination. Method:The minimum inhibitory concentrations (MICs) of Tanreqing and imipenem-cilastatin against planktonic XDPA strain isolated in clinic were determined by the broth microdilution method. The checkerboard method was used to evaluate the combination effect. The bacterial metabolic activity in mature biofilm was studied by microtiter-plate test. The destructive effect of combination drugs on dynamic biofilm was observed by using BioFlux system, and viable cells were examined by confocal laser scanning microscope (CLSM) after treatment. The scanning electron microscopy (SEM) was used for observing Pseudomonas aeruginosa and length measurement. Result:The MIC values of imipenem-cilastatin and Tanreqing were 512 mg·L^-1 and more than 16 500 mg·L^-1. The checkerboard analysis showed that Tanreqing could enhance the sensitivity of imipenem-cilastatin, while the combination drugs synergistically inhibited the growth of bacteria. Compared with the control group or the imipenem-cilastatin individual group, the combined drugs significantly reduced the amount of living bacteria in the biofilm (P<0.05,P<0.01). BioFlux results showed that the combination drugs destructed the biofilm structure and reduced the area coverage (P<0.05) by comparing with the control group or the single drug group. The results of fluorescent staining showed that the combination drug significantly inhibited the metabolic activity of bacteria in dynamic biofilm. Tanreqing inhibited bacterial division to achieve the antibacterial effect. Conclusion:Tanreqing and imipenem-cilastatin synergistically inhibit the bacterial growth in planktonic and biofilm states, and destruct biofilms.

Immunotherapy is a new strategy for cancer treatment that has the potential to treat all types of cancer. T cell immunoglobulin and mucin-domain-containing molecule-3 (TIM-3) is a key negative regulator of T cell activation. TIM-3 blockage using anti-TIM-3 monoclonal antibody therapy has a great appeal and special advantages. Nanobodies, derived from heavy chain fragment in camelid animals, are now proving clinical values in the development of antibody drugs. In this study, we have immunized camel with TIM-3 antigens and then constructed phage display library. Moreover, 29 nanobodies with different complementarity-determining regions sequences have been screened from the phage display library by phage display technology. In addition, we successfully constructed the cell line stably expressing TIM-3, and screened 10 TIM-3 nanobodies with high specificity and high affinity using flow cytometry. Our study will lay the foundation for the future screening and development of anti-TIM-3 whole humanized functional nanobody.

To investigate the effect of piperine on the disorder of glucose metabolism in the cell model with insulin resistance (IR) and explore the molecules mechanism on intervening the upstream target of AMPK signaling pathway. The insulin resistance models in HepG2 cells were established by fat emulsion stimulation. Then glucose consumption in culture supernatant was detected by GOD-POD method. Enzyme-linked immunosorbent assay(ELISA) was used to measure the levels of leptin(LEP) and adiponectin(APN) in culture supernatant; Real-time quantitative PCR was used to assess the mRNA expression of APN and LEP; and the protein expression levels of LepR, AdipoR1, AdipoR2 and the activation of AMPK signaling pathway were detected by Western blot analysis. The results showed that piperine, rosiglitazone and AMPK agonist AICAR could significantly elevate the glucose consumption in insulin resistance cell models, enhance the level of APN, promote APN mRNA transcripts and increase the protein expression of Adipo receptor. Meanwhile,AMPKα mRNA and р-AMPKα protein expressions were also increased in piperine treated cells, but both LEP mRNA expression and LepR protein expressions were decreased in piperine treated group. The results indicated that piperine could significantly ameliorate the glucose metabolism disorder in insulin resistance cell models through regulating upstream molecules (APN and LEP) of AMPK signaling pathway, and thus activate the AMPK signaling pathway.