BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are easily isolated and amplified, and facilitate the exogenous gene transfer and expression. In the human medicine, it is believed that BMSCs are ideal therapeutic cells and target cells in the gene therapy.OBJECTIVE: To investigate liposome-mediated cytosine deaminase (CD) gene transfecting rabbit BMSCs and its gene expression. DESIGN: A single sample observation. SETTING: Dalian Research and Development Center for Stem Cell and Tissue Engineering; Department of Biochemistry, College of Basic Medical Science, Dalian Medical University.MATERIALS: This study was performed at in the Dalian Research and Development Center for Stem Cell and Tissue Engineering; Department of Biochemistry, College of Basic Medical Science, Dalian Medical University from March 2006 to June 2007. New Zealand big-ear white rabbits of either gender, weighing 2.0-2.5 kg, with the age of 5 months old, were included in this study. METHODS: The CD gene was obtained from E.coli JM109 DNA by polymerase chain reaction (PCR). The fragment was cloned into pMD19-T vector. After restriction enzyme BamHI/XhoI digestion analysis and DNA sequence analysis, pIRES2-AcGFP1-CD eukaryotic expression plasmid was constructed. Meanwhile, BMSCs were harvested, cultured and identified. After enzyme digestion of eukaryotic expression plasmid, the rabbit BMSCs were transfected by Lipofectamine 2000-mediated method. Twenty-four hours after transfection, expression of green fluorescent protein was observed under an inverted fluorescent microscope. MAIN OUTCOME MEASURES: Construction of eukaryotic expression plasmid and identification of CD gene-transferred BMSCs. RESULTS: CD gene was cloned and connected to eukaryotic expression plasmid with green fluorescence. Twenty-four hours after transfecting rabbit BMSCs, it was found under an inverted microscope that under the excitation of 488 nm blue light, green fluorescence appeared in the pIRES2-AcGFP1-CD and pIRES2-AcGFP1 empty-plasmid transfected BMSCs, but not in the non-transfected ones. It indicates that CD gene successfully transferred BMSCs. CONCLUSION: BMSCs are ideal vectors in the CD gene therapy.

When the size of a neurosphere cultured in vitro reaches a certain critical value, a necrotic core will appear inside the neurosphere because of the limitation of oxygen or other nutrients transport from medium to the cells in the neurasphere. Large necrotic core will greatly reduce the expansion of NSCs. The cellular automaton (CA) model is applied in this article to model the growth of NSCs in sphere state. The appearance and enlargement of the necrotic core in a neurosphere is calculated by coupling the CA model with the nutrient diffusion analysis in bioreactors. The calculation results indicate that the culture conditions, such as seeding density, the concentration of nutrients in medium and the mass transfer coefficient between a neurosphere and medium, have some effects on the appearance of the necrotic core. However, the necrotic core mainly depends on the inner diffusion. It will certainly appear if the size of the neurosphere is large enough even the outside mass transfer is in a good condition in bioreactors. Additionally, the appearance of the necrotic core resulting from the shortage of oxygen is earlier than that caused by the limitation of glucose. And the growth of the necrotic core is very fast after its appearance, and the whole neurosphere may become necrotic. The model developed with cellular automaton and mass transfer is a good qualitative representation of NSCs growth in bioreactors.
Bioreactors ; Cell Culture Techniques ; methods ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Computer Simulation ; Models, Biological ; Neurons ; cytology ; Spheroids, Cellular ; Stem Cells ; cytology ; Tissue Engineering ; methods

OBJECTIVEHuman rhinovirus (HRV) is the most common respiratory pathogen, which causes not only acute respiratory infection and community acquired pneumonitis in children, but also asthma episode and deterioration. However, the detection of respiratory pathogen, which mainly focuses on respiratory syncytial virus, influenzaviruses A and B, parainfluenza viruses 1-3 and adenoviruses, does not include HRV yet by now in China. The absence of detection method limits the clinical understanding of HRV pathogenicity, and causes unreasonable use of antibiotics. This study aimed to establish a one-step reverse transcription (RT) PCR system for detecting specific fragment of HRV RNA, and to analyze the sequences of amplicons.

METHODSA pair of degenerate primers based on the HRV highly conserved 5'' noncoding region (NCR) were used to develop a one-step RT-PCR system for detecting HRV RNA in nasopharyngeal aspirates from 78 children with acute respiratory tract infections in the spring of 2004. All the positive PCR products were sequenced, and the sequences of the nucleotides were analyzed by using biological software and compared with those in the GeneBank.

RESULTSEleven (14.1%) of 78 samples were positive on RT-PCR, these patients were clinically diagnozed as upper respiratory tract infection (n = 7), bronchitis (n = 3) and bronchopneumonia (n = 1), respectively. Compared with the sequences of clinical and standard HRV viruses in the GeneBank, the nucleotide sequences of these 11 amplicons shared high homology of 89%-95.5%. Within the 11 amplicons, nucleotide identity varied from 75.2% to 91.8%, and the ratio of genetic variation was from 8.8% to 31.0%, which occurred in highly conserved regions and usually showed single nucleotide mutation in some special locations. These 11 amplicons attribute to the two branches of HRV cladogram, respectively. Most of mutations in highly conservative domain occurred on single ribonucleotide, mainly as transversion (C/G, A/G) and transition (T/C, A/G), some were mutations among 3 bases (A/C/G, A/T/G, A/C/T). And a few mutations involved two nearby ribonucleotide which were also found in highly conservative domain. However, ribonucleotide deletion and insertion were usually found in highly variable domain.

CONCLUSIONThe findings showed that this one-step RT-PCR system was highly specific, rapid and convenient for the detection of HRV RNA in nasopharyngeal secretions of patients with acute respiratory tract infections and that the genome of HRV viruses was highly variable.

Base Sequence ; Child ; Child, Preschool ; Female ; Genes, Viral ; Humans ; Male ; Molecular Sequence Data ; Picornaviridae Infections ; diagnosis ; virology ; RNA, Viral ; analysis ; Respiratory Tract Infections ; virology ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Rhinovirus ; genetics ; isolation & purification ; Sensitivity and Specificity ; Sequence Analysis, DNA

OBJECTIVETo analyze the clinical features and genotype in a 8-year-old boy with dyskeratosis congenita (DC).

METHODSWe reviewed the clinical data of the case and amplified 7 DC-related genes (including DKC1,TERT,TERC,TINF2,NOP10, NHP2 and WRAP53) using polymerase chain reaction for DNA sequence analysis to identify the abnormal exons.

RESULTSDNA sequence analysis showed a c.85-15T>C mutation in DKC1 gene of the patient. His mother was a carrier of the mutated gene and presented with partial clinical features such as abnormal nails.

CONCLUSIONThe mutation of c.85-15T>C in DKC1 gene was reported for the first time in China. The diagnosis of DC should be considered if a young patient presents with mucocutaneous abnormalities, bone marrow failure, cancer susceptibility and a family history of cancer. Early genetic tests can improve the diagnosis rates and reduce misdiagnosis and missed diagnosis.

Cell Cycle Proteins ; genetics ; Child ; China ; Dyskeratosis Congenita ; genetics ; pathology ; Exons ; Genotype ; Humans ; Male ; Mutation ; Nuclear Proteins ; genetics ; Polymerase Chain Reaction ; Sequence Analysis, DNA

Rhizoma phragmitis is a common Chinese herbal medicine whose effects are defined as 'clearing heat and fire, promoting fluid production to quench thirst, eliminating irritability, stopping vomiting, and disinhibiting urine'. During the Novel Coronavirus epidemic in 2020, the Weijing Decoction and Wuye Lugen Decoction, with Rhizoma phragmitis as the main herbal component, were included in The Pneumonia Treatment Protocol for Novel Coronavirus Infection (Trial Version 5) due to remarkable antiviral effects. Modern pharmacological studies have shown that Rhizoma phragmitis has antiviral, antioxidative, anti-inflammatory, analgesic, and hypoglycemic functions, lowers blood lipids and protects the liver and kidney. This review aims to provide a systematic summary of the botany, traditional applications, phytochemistry, pharmacology and toxicology of Rhizoma phragmitis.
Humans ; Plant Extracts/pharmacology* ; Rhizome ; Drugs, Chinese Herbal/therapeutic use* ; Antioxidants/therapeutic use* ; Antiviral Agents/therapeutic use* ; Medicine, Chinese Traditional ; Phytochemicals/therapeutic use* ; Ethnopharmacology ; COVID-19 Drug Treatment

OBJECTIVETo explore the role of dimethyl sulfoxide (DMSO) in the hematopoietic differentiation of human embryonic stem cells (hESCs).

METHODSThe role of DMSO in hematopoietic differentiation of hESC was investigated by using a established stepwise hematopoietic differentiation system from hESC, immunofluorescouse assay and flow cytometry. Furthermore, the window phase of DMSO action was explored by adding it to the different stage of hematopoietic differentiation.

RESULTSImmunofluorescence and flow cytometry analysis showed that DMSO significantly promoted the generation of CD43hematopoietic progenitor cells (HPC). The flow cytometry demonstrated that DMSO profoundly promoted the generation of APLNRlateral plate mesoderm cells and CD31CD34hemogenic endothelium progenitors (HEP). The addition of DMSO in the window phase of lateral plate mesoderm cell generation could markedly improve the generation of hematopoietic progenitor cells.

CONCLUSIONDMSO promotes hematopoietic differentiation of hESC through enhancing the generation of APLNR positive lateral plate mesoderm cells. The addition of DMSO in the window phase of lateral plate mesoderm cell generation can significantly improve the generation of hematopoietic progenitor cells.

Objective: To analyze the prevalence and the risk factors of fungal sepsis in 25 neonatal intensive care units (NICU) among preterm infants in China, and to provide a basis for preventive strategies of fungal sepsis. Methods: This was a second-analysis of the data from the "reduction of infection in neonatal intensive care units using the evidence-based practice for improving quality" study. The current status of fungal sepsis of the 24 731 preterm infants with the gestational age of <34⁺⁰ weeks, who were admitted to 25 participating NICU within 7 days of birth between May 2015 and April 2018 were retrospectively analyzed. These preterm infants were divided into the fungal sepsis group and the without fungal sepsis group according to whether they developed fungal sepsis to analyze the incidences and the microbiology of fungal sepsis. Chi-square test was used to compare the incidences of fungal sepsis in preterm infants with different gestational ages and birth weights and in different NICU. Multivariate Logistic regression analysis was used to study the outcomes of preterm infants with fungal sepsis, which were further compared with those of preterm infants without fungal sepsis. The 144 preterm infants in the fungal sepsis group were matched with 288 preterm infants in the non-fungal sepsis group by propensity score-matched method. Univariate and multivariate Logistic regression analysis were used to analyze the risk factors of fungal sepsis. Results: In all, 166 (0.7%) of the 24 731 preterm infants developed fungal sepsis, with the gestational age of (29.7±2.0) weeks and the birth weight of (1 300±293) g. The incidence of fungal sepsis increased with decreasing gestational age and birth weight (both P<0.001). The preterm infants with gestational age of <32 weeks accounted for 87.3% (145/166). The incidence of fungal sepsis was 1.0% (117/11 438) in very preterm infants and 2.0% (28/1 401) in extremely preterm infants, and was 1.3% (103/8 060) in very low birth weight infants and 1.7% (21/1 211) in extremely low birth weight infants, respectively. There was no fungal sepsis in 3 NICU, and the incidences in the other 22 NICU ranged from 0.7% (10/1 397) to 2.9% (21/724), with significant statistical difference (P<0.001). The pathogens were mainly Candida (150/166, 90.4%), including 59 cases of Candida albicans and 91 cases of non-Candida albicans, of which Candida parapsilosis was the most common (41 cases). Fungal sepsis was independently associated with increased risk of moderate to severe bronchopulmonary dysplasia (BPD) (adjusted OR 1.52, 95%CI 1.04-2.22, P=0.030) and severe retinopathy of prematurity (ROP) (adjusted OR 2.55, 95%CI 1.12-5.80, P=0.025). Previous broad spectrum antibiotics exposure (adjusted OR=2.50, 95%CI 1.50-4.17, P<0.001), prolonged use of central line (adjusted OR=1.05, 95%CI 1.03-1.08, P<0.001) and previous total parenteral nutrition (TPN) duration (adjusted OR=1.04, 95%CI 1.02-1.06, P<0.001) were all independently associated with increasing risk of fungal sepsis. Conclusions: Candida albicans and Candida parapsilosis are the main pathogens of fungal sepsis among preterm infants in Chinese NICU. Preterm infants with fungal sepsis are at increased risk of moderate to severe BPD and severe ROP. Previous broad spectrum antibiotics exposure, prolonged use of central line and prolonged duration of TPN will increase the risk of fungal sepsis. Ongoing initiatives are needed to reduce fungal sepsis based on these risk factors.
Infant ; Infant, Newborn ; Humans ; Birth Weight ; Intensive Care Units, Neonatal ; Retrospective Studies ; Tertiary Care Centers ; Infant, Extremely Low Birth Weight ; Gestational Age ; Infant, Extremely Premature ; Sepsis/epidemiology* ; Retinopathy of Prematurity/epidemiology* ; Bronchopulmonary Dysplasia/epidemiology*

Objectives: To investigated the safety and efficacy of treating patients with acute non-ST-segment elevation myocardial infarction (NSTEMI) and elevated levels of N-terminal pro-hormone B-type natriuretic peptide (NT-proBNP) with levosimendan within 24 hours of first medical contact (FMC). Methods: This multicenter, open-label, block-randomized controlled trial (NCT03189901) investigated the safety and efficacy of levosimendan as an early management strategy of acute heart failure (EMS-AHF) for patients with NSTEMI and high NT-proBNP levels. This study included 255 patients with NSTEMI and elevated NT-proBNP levels, including 142 males and 113 females with a median age of 65 (58-70) years, and were admitted in the emergency or outpatient departments at 14 medical centers in China between October 2017 and October 2021. The patients were randomly divided into a levosimendan group (n=129) and a control group (n=126). The primary outcome measure was NT-proBNP levels on day 3 of treatment and changes in the NT-proBNP levels from baseline on day 5 after randomization. The secondary outcome measures included the proportion of patients with more than 30% reduction in NT-proBNP levels from baseline, major adverse cardiovascular events (MACE) during hospitalization and at 6 months after hospitalization, safety during the treatment, and health economics indices. The measurement data parameters between groups were compared using the t-test or the non-parametric test. The count data parameters were compared between groups using the χ² test. Results: On day 3, the NT-proBNP levels in the levosimendan group were lower than the control group but were statistically insignificant [866 (455, 1 960) vs. 1 118 (459, 2 417) ng/L, Z=-1.25,P=0.21]. However, on day 5, changes in the NT-proBNP levels from baseline in the levosimendan group were significantly higher than the control group [67.6% (33.8%,82.5%)vs.54.8% (7.3%,77.9%), Z=-2.14, P=0.03]. There were no significant differences in the proportion of patients with more than 30% reduction in the NT-proBNP levels on day 5 between the levosimendan and the control groups [77.5% (100/129) vs. 69.0% (87/126), χ²=2.34, P=0.13]. Furthermore, incidences of MACE did not show any significant differences between the two groups during hospitalization [4.7% (6/129) vs. 7.1% (9/126), χ²=0.72, P=0.40] and at 6 months [14.7% (19/129) vs. 12.7% (16/126), χ²=0.22, P=0.64]. Four cardiac deaths were reported in the control group during hospitalization [0 (0/129) vs. 3.2% (4/126), P=0.06]. However, 6-month survival rates were comparable between the two groups (log-rank test, P=0.18). Moreover, adverse events or serious adverse events such as shock, ventricular fibrillation, and ventricular tachycardia were not reported in both the groups during levosimendan treatment (days 0-1). The total cost of hospitalization [34 591.00(15 527.46,59 324.80) vs. 37 144.65(16 066.90,63 919.00)yuan, Z=-0.26, P=0.80] and the total length of hospitalization [9 (8, 12) vs. 10 (7, 13) days, Z=0.72, P=0.72] were lower for patients in the levosimendan group compared to those in the control group, but did not show statistically significant differences. Conclusions: Early administration of levosimendan reduced NT-proBNP levels in NSTEMI patients with elevated NT-proBNP and did not increase the total cost and length of hospitalization, but did not significantly improve MACE during hospitalization or at 6 months.
Male ; Female ; Humans ; Aged ; Natriuretic Peptide, Brain ; Simendan/therapeutic use* ; Non-ST Elevated Myocardial Infarction ; Heart Failure/drug therapy* ; Peptide Fragments ; Arrhythmias, Cardiac ; Biomarkers ; Prognosis