OBJECTIVE To establish a preparation system for megakaryocytes(MKs)derived from human embryonic stem cells(hESCs)and MK microparticles(MKMPs),and to assess the pro-angio-genic efficiency of these microparticles.METHODS ①hESCs were induced to mesodermal progenitor cells via monolayer culture with the first-stage induction medium for 2 days before the cells were induced to hemogenic endothelial/hematopoietic progenitor cells by culturing with the second-stage induction medium for another 3 days.Then,the cells were dissociated into single cells,seeded into the third-stage induction medium,and cultured using the suspension method for 8 days to obtain MKs.The specific characters of differentiated cells were identified through morphological observation and flow cytometry before stage-specific marker proteins in different periods were analyzed[hESCs:TRA-1-60,sialyl glycolipid stage-specific embryonic antigen4(SSEA4)];mesodermal progenitor cells:brachyury;hemogenic endothelial/hematopoietic progenitor cells:CD34,CD43;MKs:CD41a,CD42b),and immu-nofluorescence staining[β1-tubulin,von Willebrand factor(VWF)],[friend leukemia integration 1(FLI1),CD42].② MKMP collection and verification:MKMPs were collected via differential centrifugation.The concentration and size of these MKMPs were determined by nanoparticle tracking analysis(NTA),and both the morphology and ultrastructure were examined by transmission electron microscopy(TEM).Besides,the MKMPs-specific proteins[CD41,tumor susceptibility gene 101(TSG101)and CD9]were detected by Western blotting analysis.③ Biological function of MKMPs:MKMPs were stained with CD41a-PE antibodies and co-cultured with human umbilical veinvascular endothelial cells(HUVECs)labeled by CD34-APC for 3 h.Live-cell immunofluorescence was employed to find out whether HUVECs could absorb MKMPs.To find out whether MKMPs could affect the role of HUVECs in angio-genesis and cell migration,platelet microvesicles(PMPs)were used as positive controls.The experi-mental groups were added with different concentrations of microparticles(1,5,10 and 20 mg·L-1)while the control group was given no microparticles(0 mg·L-1).The number of nodes that formed the lumen after 5 h of incubation in Matrigel was counted,and the size of healing of the scratch area was analyzed after 6 h.To elucidate the mechanism through which MKMPs impacted angiogenesis,ELISA was used out to quantitatively detect the concentration of proteins in microparticles.RESULTS ① A three-stage differentiation cultural system was established to develop hESCs into MKs.Flow cytometry revealed progressive loss of pluripotency markers SSEA4 and TRA-1-60,while the mesodermal progenitor marker brachyury peaked at d 2.Subsequently,hemogenic endothelial/hematopoietic progenitor markers CD34 and CD43 emerged at d 5,followed by megakaryocytic markers CD41a and CD42b at d 13.Immunofluorescent images further demonstrated that MKs expressed specific proteins CD42,β1-tubulin,von VWF and FLI1 at d 13.②Microparticles were collected via differential centrifuga-tion.Transmission electron microscopy revealed that their substructure exhibited a typical double-layered membrane.Nanoparticle tracking analysis indicated that the size was(164.3±14.0)nm.The result of WB demonstrated that the microparticles expressed specific markers,including TSG101,CD9 and CD41.③ MKMPs were absorbed after being co-cultured with HUVECs for 3 h and enhanced the ability of HUVECs to form tubes and migrate.Notably,the treatment of 5 mg·L-1 MKMPs was more effective than 5 mg·L-1 PMPs treatment.The results of ELISA showed that the content of VEGF from MKMPs was higher than from PMPs,which may be the key factor in regulating endothelial biological function.CONCLUSION MKs derived from hESCs can generate functional microparticles which can promote angiogenesis.

OBJECTIVE To establish a preparation system for megakaryocytes(MKs)derived from human embryonic stem cells(hESCs)and MK microparticles(MKMPs),and to assess the pro-angio-genic efficiency of these microparticles.METHODS ①hESCs were induced to mesodermal progenitor cells via monolayer culture with the first-stage induction medium for 2 days before the cells were induced to hemogenic endothelial/hematopoietic progenitor cells by culturing with the second-stage induction medium for another 3 days.Then,the cells were dissociated into single cells,seeded into the third-stage induction medium,and cultured using the suspension method for 8 days to obtain MKs.The specific characters of differentiated cells were identified through morphological observation and flow cytometry before stage-specific marker proteins in different periods were analyzed[hESCs:TRA-1-60,sialyl glycolipid stage-specific embryonic antigen4(SSEA4)];mesodermal progenitor cells:brachyury;hemogenic endothelial/hematopoietic progenitor cells:CD34,CD43;MKs:CD41a,CD42b),and immu-nofluorescence staining[β1-tubulin,von Willebrand factor(VWF)],[friend leukemia integration 1(FLI1),CD42].② MKMP collection and verification:MKMPs were collected via differential centrifugation.The concentration and size of these MKMPs were determined by nanoparticle tracking analysis(NTA),and both the morphology and ultrastructure were examined by transmission electron microscopy(TEM).Besides,the MKMPs-specific proteins[CD41,tumor susceptibility gene 101(TSG101)and CD9]were detected by Western blotting analysis.③ Biological function of MKMPs:MKMPs were stained with CD41a-PE antibodies and co-cultured with human umbilical veinvascular endothelial cells(HUVECs)labeled by CD34-APC for 3 h.Live-cell immunofluorescence was employed to find out whether HUVECs could absorb MKMPs.To find out whether MKMPs could affect the role of HUVECs in angio-genesis and cell migration,platelet microvesicles(PMPs)were used as positive controls.The experi-mental groups were added with different concentrations of microparticles(1,5,10 and 20 mg·L-1)while the control group was given no microparticles(0 mg·L-1).The number of nodes that formed the lumen after 5 h of incubation in Matrigel was counted,and the size of healing of the scratch area was analyzed after 6 h.To elucidate the mechanism through which MKMPs impacted angiogenesis,ELISA was used out to quantitatively detect the concentration of proteins in microparticles.RESULTS ① A three-stage differentiation cultural system was established to develop hESCs into MKs.Flow cytometry revealed progressive loss of pluripotency markers SSEA4 and TRA-1-60,while the mesodermal progenitor marker brachyury peaked at d 2.Subsequently,hemogenic endothelial/hematopoietic progenitor markers CD34 and CD43 emerged at d 5,followed by megakaryocytic markers CD41a and CD42b at d 13.Immunofluorescent images further demonstrated that MKs expressed specific proteins CD42,β1-tubulin,von VWF and FLI1 at d 13.②Microparticles were collected via differential centrifuga-tion.Transmission electron microscopy revealed that their substructure exhibited a typical double-layered membrane.Nanoparticle tracking analysis indicated that the size was(164.3±14.0)nm.The result of WB demonstrated that the microparticles expressed specific markers,including TSG101,CD9 and CD41.③ MKMPs were absorbed after being co-cultured with HUVECs for 3 h and enhanced the ability of HUVECs to form tubes and migrate.Notably,the treatment of 5 mg·L-1 MKMPs was more effective than 5 mg·L-1 PMPs treatment.The results of ELISA showed that the content of VEGF from MKMPs was higher than from PMPs,which may be the key factor in regulating endothelial biological function.CONCLUSION MKs derived from hESCs can generate functional microparticles which can promote angiogenesis.

Objective To validate the performance of droplet digital polymerase chain reaction (ddPCR) reagents using clinical and standard strains, and to evaluate the effectiveness and practicability of ddPCR technology in clinical applications. Methods The concordance rate, specificity, precision, and lower limit of detection of the ddPCR kit were validated using clinical and standard strains. Blood samples from 74 patients with suspected bloodstream infections were collected, and both ddPCR and blood culture methods were used to determine the pathogens in the patient's blood samples. Results The average detection time of ddPCR for pathogens of bloodstream infection was 3.5 hours, which was able to complete the detection of over a dozen common pathogens simultaneously. The concordance rate, specificity, precision, and lower limit of detection of the ddPCR kit for bloodstream infection pathogens all met clinical requirements. Among the 74 patients with suspected bloodstream infections, the positive detection rate using the ddPCR method was 64.86%, while was 40.54% using blood culture, with a statistically significant difference (P < 0.05). Conclusion The method of ddPCR can detect common pathogens of bloodstream infections rapidly, efficiently, and in large quantities, and its main detection performance can meet clinical needs. The combination of blood culture and ddPCR technology is conductive to early and rapid diagnosis of clinical bloodstream infection patients.

Objective To construct a model of heart failure risk prediction based on four machine learning algo-rithms in order to support early diagnosis and intervention.Methods After reviewing the heart failure dataset pub-lished on the Kaggle community,feature selection was used to select relevant factors related to heart failure as pre-dictive indicators.Four machine learning algorithms,namely logistic regression,support vector machine,random forest,and XGBoost were selected to establish predictive models.Compared and analyzed its accuracy,precision,recall,F1 score and area under the ROC curve(AUC)to verify the performance of the model.Results The study analyzed 11 features of 918 patients with heart failure and selected 10 feature factors for modeling.After optimizing the hyper-parameters through grid search,the XGBoost model performed the best,with accuracy,precision,recall,and f1_score and AUC values were 87.5%,90.38%,89.71%,90.04%and 0.93,respectively.In addition,data analysis showed that exercise ST slope,chest pain type,and exercise induced angina were main influencing factors for heart failure.Conclusions The XG Boost model has the best predictive tool for heart failure,and machine learning algorithms may support early prevention,early diagnosis as well as control of heart failure.

Objective To observe the consistency of 5.0T and 1.5T MR spectroscopy(MRS)for quantitating proton density fat fraction(PDFF)of liver.Methods Lipid emulsion models with lipid content of 0,5％,10％,15％,20％,25％and 30％were prepared.1H-MRS were collected using 5.0T and 1.5T MR scanners,respectively,and PDFF were obtained with jMRUI software.Totally 23 people,including 11 cases of fatty liver and 12 healthy adults were prospectively collected,and volume of interest(VOI)in the liver were selected to acquire 1H-MRS,and PDFF were obtained with jMRUI software and corresponding workstation,respectively.The consistencies of PDFF measured with different methods were analyzed.Results PDFF of lipid emulsion models with lipid content of 0,5％,10％,15％,20％,25％and 30％measured with jMRUI software and workstations based on 5.0T and 1.5T 1H-MRS all had good consistencies and being positively correlated,so were PDFF of liver tissue measured with jMRUI software and workstations based on 5.0T and 1.5T 1H-MRS.Conclusion 5.0T and 1.5T 1H-MRS had good consistency for quantitating liver PDFF.Measuring liver PDFF with workstation in clinical practice was helpful to simplifying workflow.

Objective:To observe the effect of high expression of polypyrimidine tract-binding protein-associated splicing factor (PSF) on low concentration of 4-hydroxynonenal (4-HNE) induced human retinal microvascular endothelial cells (HRMECs), and explore the possible mechanism.Methods:The HRMECs cultured in vitro were divided into 4-HNE treated group, PSF overexpression group combined with 4-HNE group (PSF+4-HNE group), PSF overexpression+ML385 treatment combined with 4-HNE group (PSF+ML385+4-HNE group), and 4-HNE induced PSF overexpression group with LY294002 pretreatment (LY294002+4-HNE+PSF group). Cell culture medium containing 10 μmmol/L 4-HNE was added into 4-HNE treatment group, PSF+4-HNE group, PSF+ML385+4-HNE group for 12 hours to stimulate oxidative stress. 1.0 μg of pcDNA-PSF eukaryotic expression plasmid were transfected into PSF+4-HNE group and PSF+ML385+4-HNE group to achieve the overexpression of PSF. Also cells were pretreated with ML385 (5 μmol/L) for 48 hours in the PSF+ML385+4-HNE group, meanwhile within the LY294002+4-HNE+PSF group, after pretreatment with LY294002, cells were treated with plasmid transfection and 4-HNE induction. Transwell detects the migration ability of PSF to HRMECs. The effect of PSF on the lumen formation of HRMECs was detected by using Matrigel in vitro three-dimensional molding method. Flow cytometer was used to detect the effect of PSF overexpression on reactive oxygen (ROS) level in HRMECs. Protein immunoblotting was used to detect the relative expression of PSF, nuclear factor E2 related factor 2 (Nrf2), heme oxygenase-1 (HO-1) protein, and phosphoserine threonine protein kinase (pAkt) protein. The comparison between the two groups was performed using a t-test. Results:The number of live cells, migrating cells, and intact lumen formation in the 4-HNE treatment group and the PSF+4-HNE group were 1.70±0.06, 0.80±0.13, 24.00±0.58, 10.00±0.67, and 725.00±5.77, 318.7±12.13, respectively. There were significant differences in the number of live cells, migrating cells, and intact lumen formation between the two groups ( t=12.311, 15.643, 17.346; P<0.001). The results of flow cytometry showed that the ROS levels in the 4-HNE treatment group, PSF+4-HNE group, and PSF+ML385+4-HNE group were 816.70±16.67, 416.70±15.44, and 783.30±17.41, respectively. There were statistically significant differences between the two groups ( t=16.311, 14.833, 18.442; P<0.001). Western blot analysis showed that the relative expression levels of pAkt, Nrf2, and HO-1 proteins in HRMECs in the 4-HNE treatment group, PSF+4-HNE group and LY294002+4-HNE+PSF group were 0.08±0.01, 0.57±0.04, 0.35±0.09, 0.17±0.03, 1.10±0.06, 0.08±0.11 and 0.80±0.14, 2.50±0.07, 0.50±0.05, respectively. Compared with the PSF+4-HNE group, the relative expression of pAkt, Nrf2, and HO-1 proteins in the LY294002+4-HNE+PSF group decreased significantly, with significant differences ( t=17.342, 16.813, 18.794; P<0.001). Conclusion:PSF upregulates the expression of HO-1 by activating the phosphatidylinositol 3 kinase/Akt pathway and inhibits cell proliferation, migration, and lumen formation induced by low concentrations of 4-HNE.

Objective:To explore the necessity and feasibility of clinical nutrition pharmacist-involved consultation, and evaluate the impact on clinical outcome.Methods:Patients who received the nutritional consultation involving pharmacists at Shanxi Bethune Hospital between 2019 and 2021 were retrospectively enrolled. Patient clinical characteristics, distribution, consultation purpose and nutrition management regimen, and clinical outcomes were collected and analyzed.Results:There was increasing utilization of nutrition pharmacist-involved consultations and referrals to pharmacy clinic over the three years. An average of 95.3% of consultation advices were accepted. Over 90% of patients who were scored 3-6 points with Nutritional Risk Screening 2002 benefited from such consultations. Patients with tumors, elderly patients, and patients with digestive system disorders showed the most benefit from nutritional consultations. The purpose of consultation was focused on the development of individualized nutritional management plans, which brought about improvement in patient's nutritional indicators.Conclusion:There is a strong demand for nutritional pharmacy consultation in large-scale comprehensive hospitals, and the standardized consultation workflow paves the way for comprehensive and individualized nutrition therapy.

Objective:To explore the effect of long-chain fat emulsion in parenteral nutrition therapy on the perioperative nutritional status of patients with low rectal cancer.Methods:A total of 204 patients who underwent rectal cancer surgery in the Third Hospital of Shanxi Medical University from January 2017 to June 2020 were retrospectively analyzed. The patients were divided into two groups according to the specific nutritional treatment methods, 100 cases in the study group used long-chain fat emulsion for parenteral nutrition support, and 104 cases in the control group used medium- and long-chain fat emulsion injection. After admission, the nutritional status of patients were evaluated according to the results of Scored Patient-Generated Subjective Global Assessment (PG-SGA) and related laboratory tests. At 7th day before the operation, the patients were treated with nutrition and electrolyte support. Parenteral nutrition and enteral nutrition combined treatment and early enteral nutrition were given after the operation. The albumin, prealbumin, retinol-binding protein, total cholesterol and body mass index (BMI) at 7th day before the operation, 1st day after the operation and 7th day after the operation and the patient's first exhaust time after surgery, occurrence of postoperative complications, postoperative fever and total hospital stay were recorded and compared between the two groups.Results:Postoperative first exhaust time [(42±11) h vs. (54±10) h], fever time [(48±8) h vs. (57±7) h], total hospital stay [(16.0±0.7) d vs. (18.0±0.9) d)], resting energy expenditure at the 7th day after surgery [(5 326±589) kJ/d vs. (5 840±599) kJ/d] and total cholesterol at the 7th day after surgery [(4.8±0.3) mmol/L vs. (5.0± 0.4) mmol/L] in the study group were lower than those in the control group, and albumin [(33±3) g/L vs. (28± 3) g/L], prealbumin [(0.189±0.041) g/L vs. (0.164±0.037) g/L] and retinol-binding protein [(0.039±0.016) g/L vs. (0.032±0.013) g/L] at the 7th day after surgery in the study group were higher than those in the control group, and the differences between the two groups were statistically significant (all P < 0.05). There was no statistical difference in other detection indexes between the two groups (all P > 0.05). Conclusion:The use of long-chain fat emulsion in low rectal cancer patients with malnutrition during the perioperative period may be more conducive to the recovery of the body.

In recent years, with the changes in living standards and dietary structure, the incidence of non-alcoholic fatty liver disease has been increasing year by year in China, and the incidence rate in the general population is as high as 29.81%. An increasingly epidemiological evidence suggests that non-alcoholic fatty liver disease has become one of the causes of increasing liver cirrhosis and liver cancer. However, its etiology and pathogenesis are complex and have not yet been fully elucidated. Therefore, establishing an appropriate non-alcoholic fatty liver disease animal models for pre-clinical research is essential to elucidate its pathogenesis. This article summarizes the latest research progress of non-alcoholic fatty liver disease animal models, which are common at home and abroad in recent years.

Objective: To investigate the effects of modified acidic fibroblast growth factor (MaFGF) mediated by nanoliposomes combined with ultrasound-targeted microbubble destruction (UTMD) on left ventricular systolic function in early diabetes mellitus(DM) rats. Methods: The nanoliposomes containing MaFGF(MaFGF-nlip) were prepared by reverse phase evaporation method. Among 60 male Sprague Dawley (SD) rats, 50 rats were randomly selected and were induced to be DM models by streptozotocin(STZ) through intraperitoneal injecting, the other 10 rats as control group. Then DM rats were randomly divided into 4 groups: DM model group, MaFGF solution group, MaFGF-nlip group and MaFGF-nlip+ UTMD group. After the successful induction of DM model, the intervention was performed twice a week.After 12 weeks of intervention, all rats underwent conventional echocardiography and velocity vector imaging (VVI). Left ventricular ejection fraction (LVEF) and left ventricular fraction shortening(LVFS) were measured by conventional echocardiography. The mean peak systolic radial velocity (Vs), radial strain (Sr) and radial strain rate (SRr) of six walls at the papillary muscle level were measured in left ventricular short-axis view by VVI. At last, myocardial tissue of all rats were stained with Sirius red to evaluate myocardial interstitial fibrosis. The level of myocardial apoptosis was evaluated by TUNEL staining, and the changes of myocardial ultrastructure were observed by transmission electron microscopy. Results: The prepared MaFGF-nlip were more rounded, evenly dispersed, and of good stability and high encapsulation efficiency. Twelve weeks later after intervention, LVEF, LVFS, Vs, Sr and SRr in the DM model group were significantly lower than those in the control group (all P<0.05). LVEF, LVFS, Vs, Sr and SRr in the MaFGF-nlip+ UTMD group were significantly higher than those of the DM model group and other intervention groups (all P<0.05). The results of Sirius red staining and Tunel staining showed that CVF and AI in the DM model group were significantly higher than those in the control group (all P<0.05). For MaFGF-nlip+ UTMD group, CVF and AI were significantly decreased compared with the DM model group and other intervention groups(all P<0.05). According to the results of transmission electron microscopy, compared with the DM model group, the improvement of myocardial ultrastructure was the most obvious in the MaFGF-nlip+ UTMD group. Conclusions MaFGF delivered by using nanoliposomes combined with UTMD can improve the left ventricular systolic function in diabetic rats by inhibiting the myocardium cardiac fibrosis and reducing the cardiomyocyte apoptosis.