Expression and relationship of miR-379 and human kinesin like family member 4A in triple-negative breast cancer / 中国医师杂志

Objective:To analyze the expression and relationship of miR-379 and human kinesin family member 4A (KIF4A) in triple-negative breast cancer (TNBC).Methods:A total of 52 patients diagnosed with TNBC in breast department at Puji Branch of Dongguan People′s Hospital between January 2016 and November 2019 were retrospectively selected as the study group. Meanwhile, 70 patients with non-triple-negative breast cancer (NTNBC) diagnosed in the same period were selected as the control group. The expression levels of miR-379 and KIF4A in both groups were detected. The receiver operating characteristic (ROC) curve was drawn, and the area under the curve (AUC) of miR-379 and KIF4A predicting TNBC was calculated; The relationship between the expression levels of miR-379 and KIF4A and clinicopathological parameters, and the correlation between the expression level of miR-379 and KIF4A were analyzed.Results:The expression level of KIF4A in study group was higher than that in control group [(6.93±0.43) vs (3.75±0.25), P<0.05], and the expression level of miR-379 in study group was lower than that in control group [(0.54±0.17) vs (0.87±0.32), P<0.05]. MiR-379 combined with KIF4A expression had the greatest diagnostic value for TNBC: AUC 0.823 (95% CI: 0.730- 0.917), specificity 0.785, sensitivity 0.950; Univariate analysis showed that there were significant difference in the expression of miR-379 in TNBC patients with different clinical stages, tumor diameter, and lymph node metastasis (all P<0.05); There were significant difference in the expression of KIF4A in TNBC patients with different clinical stages and tumor diameter (all P<0.05). Pearson correlation analysis showed that miR-379 expression was negatively correlated with KIF4A expression in TNBC ( r=-0.349, P<0.05). Conclusions:The expression of miR-379 is down-regulated, while the expression of KIF4A is up-regulated in patients with TNBC. The two are related to poor clinicopathological characteristics, which indicates that they can be used for condition evaluation of the patients.

Exploration of clinicians′ practice competency evaluation based on medical record homepage data / 中华医院管理杂志

The professional title evaluation of health professionals needs to highlight the clinical performance and actual contribution, and make full use of the information system of medical and health institutions to collect relevant data as an important basis for such evaluation.Based on this, the project team innovatively developed a " clinical work data extraction system" , to extract and calculate the performance indexes of clinicians using data from homepages of medical records. Meanwhile, the team established a reference scale based on the data in the hospital quality monitoring system; developed a " health workers evaluation data platform" , visually presenting the comparison results between the clinical work performance evaluation data of a clinician, and the reference scale and the data of other applicants. In the 2021 annual evaluation of senior professional titles among some medical institutions directly under the National Health Commission and such provinces as Sichuan, Shandong and Chongqing, this method was used to extract homepage data of medical records of 7 833 applicants from 39 medical specialties in 1 416 medical institutions, and finally 6 093 people (77.79%) completed the calculation of clinical work evaluation index data. The initial application results showed that the evaluation of senior clinicians′ professional competence based on homepage data of the medical record was feasible in the senior professional title evaluation of various medical institutions at all levels equipped with the electronic medical record database system, and could effectively present the performance level and actual contribution of the applicant.

Rapid generation of double-layer emulsion droplets based on microfluidic chip / 生物工程学报

In vitro compartmentalization (IVC) links genotype and phenotype by compartmentalizing individual genes (including expression system) or cells into a micro-droplet reaction region. Combined with fluorescence-activated cell sorting (FACS), it can detect and separate single droplets in ultra-high throughput. IVC-FACS screening method has been widely used in protein engineering, enzyme directed evolution, etc. However, it is difficult to control the homogeneity of droplet size by mechanical dispersion method in previous studies, which seriously affects the quantitative detection of droplets and reduces the efficiency and accuracy of this screening method. With the rapid development of microfluidic chip manufacturing technology, the microfluidic chip-based methods for droplet generation are becoming more efficient and controllable. In this study, firstly, the water-in-oil (W/O) single-layer droplet generation chip was used to prepare single-layer monodisperse W1/O droplets at a high generation frequency, and then the W1/O droplets were reinjected into water-in-oil-in-water (W/O/W) double-layer droplet generation chip to prepare uniform W1/O/W2 double-layer emulsion droplets. By optimizing the flow rate and ratio of the oil and water phases, a single-layer micro-droplet can be generated with a diameter range from 15.4 to 23.2 μm and remain stable for several days under normal incubation. Then the single-layer droplets were reinjected into the double emulsion generation chip. By adjusting the flow rate of drop phase, oil phase and water phase, the double-layer emulsion droplets with a diameter range from 30 to 100 μm at a rate of 1 000 droplets/s could be obtained. Escherichia coli embedded in the double-layer emulsion droplets could be cultured and induced for protein expression. This study lays a foundation for the establishment of a high-throughput screening method based on the droplet and flow cytometry.

Development and application of a droplet-based microfluidic high-throughput screening of Pichia pastoris / 生物工程学报

Pichia pastoris is one of the most convenient and widely used heterologous protein expression systems. To further improve its ability to express heterologous proteins, we developed a high-throughput P. pastoris screening method based on droplet microfluidic and demonstrated the method by screening and obtaining mutants with enhanced xylanase expression and secretion abilities. We used PCR (Polymerase Chain Reaction) amplification to obtain a fusion fragment of xylanase xyn5 gene and green fluorescent protein gfp gene, and cloned this fragment into pPIC9K, the expression vector of Pichia pastoris, to construct the plasmid pPIC9K-xyn5-gfp that recombined the DNA fragments of xylanase and green fluorescent protein. After this plasmid entered P. pastoris GS115 by electroporation, the P. pastoris SG strain that could express xylanase and green fluorescent protein was obtained. The above-said strains were then mutagenized by atmospheric room temperature plasma and subsequently encapsulated to form single-cell droplets. After 24-hour cultivation of the droplets, microfluidic screening was carried out to obtain the mutant strain with high xylanase expression for further construction and screening of the next mutagenesis library. After five rounds of droplet microfluidic screening, a highly productive strain P. pastoris SG-m5 was obtained. The activity of the expressed xylanase was 149.17 U/mg, 300% higher than that of those expressed by the original strain SG. This strain's ability to secrete heterologous protein was 160% higher than that of the original strain. With a screening throughput of 100 000 strains per hour, the high-throughput P. pastoris screening system based on single-cell droplet microfluidic developed by the present study screens a library with million strains in only 10 hours and consumes only 100 μL of fluorescent reagent, thus reducing the reagent cost by millions of times compared with the traditional microplate screening and more importantly, providing a novel method to obtain P. pastoris with high abilities to express and secret heterologous proteins by efficient and low-cost screening.

Micro-droplet characterization and its application for amino acid detection in droplet microfluidic system / 生物工程学报

Recently, the droplet microfluidic system attracts interests due to its high throughput and low cost to detect and screen. The picoliter micro-droplets from droplet microfluidics are uniform with respect to the size and shape, and could be used as monodispensed micro-reactors for encapsulation and detection of single cell or its metabolites. Therefore, it is indispensable to characterize micro-droplet and its application from droplet microfluidic system. We first constructed the custom-designed droplet microfluidic system for generating micro-droplets, and then used the micro-droplets to encapsulate important amino acids such as glutamic acid, phenylalanine, tryptophan or tyrosine to test the droplets' properties, including the stability, diffusivity and bio-compatibility for investigating its application for amino acid detection and sorting. The custom-designed droplet microfluidic system could generate the uniformed micro-droplets with a controllable size between 20 to 50 microm. The micro-droplets could be stable for more than 20 h without cross-contamination or fusion each other. The throughput of detection and sorting of the system is about 600 micro-droplets per minute. This study provides a high-throughput platform for the analysis and screening of amino acid-producing microorganisms.

Effects of platelet factor 4 on expression of bone marrow heparan sulfate in syngenic bone marrow transplantation mice / 华中科技大学学报（医学）（英德文版）

To explore the effects of platelet factor 4(PF4) on hematopoietic reconstitution and its mechanism in syngenic bone marrow transplantation (BMT). The syngenic BMT mice models were established. 20 and 26 h before irradiation, the mice were injected 20 micrograms/kg PF4 or PBS twice into abdominal cavity, then the donor bone marrow nuclear cells (BMNC) were transplanted. On the 7th day, spleen clone forming units (CFU-S) were counted. On the 7th, 14th and 21st day after BMT, the BMNC and megakaryoryocytes in bone marrow tissue were counted and the percentage of hematopoietic tissue and expression level of heparan sulfate in bone marrow tissue were assessed. In PF4-treated groups, the CFU-S counts on the 7th day were higher than those in BMT groups after BMT. The BMNC and megakaryoryocyte counts and the percentage of hematopoietic tissue and heparan sulfate expression level were higher than those in BMT group on the 7th, 14th and 21st day after BMT (P < 0.01 or P < 0.05). PF4 could accelerate hematopoietic reconstitution of syngenic bone marrow transplantation. The promotion of the heparan sulfate expression in bone marrow may be one of mechanisms of PF4.

Detection of Estrogen Receptor in Breast Cancer by Flow Cytometry / 中国医师杂志

Objective To detect estrogen receptor and DNA index in breast cancer by flow cytometry. Methods 32 cases of fresh specimens of breast cancer resected by operation were used in this study. Single cells suspension was prepared from those specimens, and estrogen receptor expression was analyzed by flow cytometry. At the same time, the status of ploid and S phase cell ratio were determined. Results Flow cytometry analysis could measure the expression level of estrogen receptor in the fresh breast cancer specimens, which was more sensitive and needed less volume of tumor tissue compared with immuohistochemical method. The information on the status of ploid and S phase cell ratio were simultaneously obtained. Conclusion The detection of estrogen receptor in breast cancers by flow cytometry was more helpful for evaluating prognosis and selecting treatment.

The Study of Cell-cycle Specificity of Tamoxifen-induced Apoptosis in the ER(+) Breast Cancer Cells / 中国医师杂志

Objective To study the cell-cycle specificity of tamoxifen-induced apoptosis in the ER(+) breast cancer cells in vitro and in vivo. Methods ER(+) MCF-7 cell line (in vitro) and primary cultured ER(+) breast cancer cells (in vivo) were treated with tamoxifen, and the cell cycle specificity of cell apoptosis and the apoptotic rate were dertermined by flow cytometry. Results Both MCF-7 and primary cultured breast cancer cells were induced to apoptosis with G 0/G 1 phase specificity by tamoxifen. And the apoptotic rate in MCF-7 was higher than that in primary cultured breast cancer cells. Conclusion Tamoxifen could induced G 0/G 1 phase specific apoptosis in the ER(+) breast cnacer cells, and tamoxifen-induced apoptotic rate was higher in vitro than in vivo.