CRISPR/Cas(the clustered regularly interspaced short palindromic repeats-CRISPR associated)system exists in most bacteria and all archaea. It is an important strategy for bacteria and archaea to resist foreign nucleic acid invasion and use for self-defense. The CRISPR/Cas system is a simple, fast, and specific diagnostic tool, which is widely used in agriculture, industry, animal husbandry, and medicine. This article mainly introduces and discusses recently advantages and limitations of biosensors combining CRISPR/Cas system with fluorescence, visualization and surface enhanced raman related technologies, as well as future research directions.
Animals ; CRISPR-Cas Systems ; Bacteria/genetics* ; Archaea

OBJECTIVES: To investigate the clinical treatment outcomes and the changes of the outcomes over time in extremely preterm twins in Guangdong Province, China. METHODS: A retrospective analysis was performed for 269 pairs of extremely preterm twins with a gestational age of <28 weeks who were admitted to the department of neonatology in 26 grade A tertiary hospitals in Guangdong Province from January 2008 to December 2017. According to the admission time, they were divided into two groups: 2008-2012 and 2013-2017. Besides, each pair of twins was divided into the heavier infant and the lighter infant subgroups according to birth weight. The perinatal data of mothers and hospitalization data of neonates were collected. The survival rate of twins and the incidence rate of complications were compared between the 2008-2012 and 2013-2017 groups. RESULTS: Compared with the 2008-2012 group, the 2013-2017 group (both the heavier infant and lighter infant subgroups) had lower incidence rates of severe asphyxia and smaller head circumference at birth (P<0.05). The mortality rates of both of the twins, the heavier infant of the twins, and the lighter infant of the twins were lower in the 2013-2017 group compared with the 2008-2012 group (P<0.05). Compared with the 2008-2012 group, the 2013-2017 group (both the heavier infant and lighter infant subgroups) had lower incidence rates of pulmonary hemorrhage, patent ductus arteriosus (PDA), periventricular-intraventricular hemorrhage (P-IVH), and neonatal respiratory distress syndrome (NRDS) and a higher incidence rate of bronchopulmonary dysplasia (P<0.05). CONCLUSIONS There is a significant increase in the survival rate over time in extremely preterm twins with a gestational age of <28 weeks in the 26 grade A tertiary hospitals in Guangdong Province. The incidences of severe asphyxia, pulmonary hemorrhage, PDA, P-IVH, and NRDS decrease in both the heavier and lighter infants of the twins, but the incidence of bronchopulmonary dysplasia increases. With the improvement of diagnosis and treatment, the multidisciplinary collaboration between different fields of fetal medicine including prenatal diagnosis, obstetrics, and neonatology is needed in the future to jointly develop management strategies for twin pregnancy.
Bronchopulmonary Dysplasia/epidemiology* ; Female ; Gestational Age ; Humans ; Infant ; Infant, Extremely Premature ; Infant, Newborn ; Pregnancy ; Respiratory Distress Syndrome, Newborn/epidemiology* ; Retrospective Studies ; Treatment Outcome

OBJECTIVE: To detect the expression of CRLF2 in bone marrow mononuclear cells from children with newly diagnosed acute lymphoblastic leukemia(ALL) and to explore its clinical significance in pediatric ALL. METHODS: A total of 218 children with newly diagnosed ALL who achieveal the complete remission and had the complete follow-up information were selected, and the expression level of CRLF2 in bone marrow mononuclear cells of these children was detected by real-time fluorescent quantitative PCR, and the significance of CRLF2 expression level in clinical prognosis of ALL children was analyzed by using statistical method. RESULTS: 28 cases in 218 children with complete data showed high expression of CRLF2. The cumulative recurrence rate in the CRLF2 high expression group was significantly higher than that in the low expression group (53.6% vs 12.6%) (P＜0.01). The predicted 5-year recurrence-free survival rate (RFS) of ALL children with CRLF2 high expression was significantly higher than that of low expression group (P＜0.01). There was no significant difference in the predicted 5-year RFS between ALL children with CRLF2 low and high expression in the standard-risk(SR) group (P＞0.05). The predicted 5-year RFS of ALL children with CRLF2 low expression was higher than that of ALL children with CRLF2 high expression in the intermediate-risk (IR) and high-risk (HR) groups. (P＜0.05). Cox analysis showed that CRLF2 high expression is an independent risk factor for the relapse of children with ALL. CONCLUSION The recurrence rate of pediatric ALL with CRLF2 high expression is high, and CRLF2 high expression is an important prognostic factor for high risk of relapse in ALL children with IR and HR. It is necessary to use CRLF2 expression as an indicator of risk stratification in pediatric ALL.
Bone Marrow ; Child ; Humans ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Prognosis ; Receptors, Cytokine ; metabolism ; Recurrence ; Risk Factors

OBJECTIVETo study the effects of minimal residual disease (MRD) level on day 33 of remission induction and IKZF1 genotype on the survival of children with B-lineage acute lymphoblastic leukemia (B-ALL).

METHODSA total of 152 children with newly-diagnosed B-ALL who had complete remission after the first cycle of the chemotherapy and had complete follow-up information were enrolled in this study. According to the MRD detection by flow cytometry on day 33 of remission induction, they were divided into three groups: standard-risk (SR) group (MRD <10; n=60), intermediate-risk (IR) group (10≤ MRD <10; n=55), and high-risk (HR) group (MRD ≥10; n=37). Nested RT-PCR was used to determine the IKZF1 genotype of all children before chemotherapy. The effects of MRD level on day 33 of remission induction and IKZF1 genotype on the recurrence-free survival (RFS) of children with B-ALL were analyzed.

RESULTSThere were 7 common IKZF1 subtypes in all the 152 children with B-ALL: IK1, IK2/3, IK4, IK6, IK8, IK9, and IK10. Of the 152 children, 130 had functional subtypes of IKZF1 and 22 had non-functional subtypes of IKZF1. During the follow-up period, relapse occurred in 26 (17%) children, and the recurrence rate was highest in the HR group (P<0.05). However, there was no significant difference in the recurrence rate between the SR group and the IR group (P>0.05). The cumulative recurrence rate of the children with non-functional subtypes of IKZF1 was significantly higher than that of those with functional types of IKZF1 (P<0.01). The predicted 5-year RFS rates in the SR, IR, and HR groups were (94.2±2.9)%, (86.7±3.8)%, and (56.2±4.5)% respectively (P<0.05). The 5-year RFS rate of the children with functional subtypes of IKZF1 was significantly higher than that of those with non-functional subtypes of IKZF1 (P<0.01). There was no significant difference in the predicted 5-year RFS rate between the children with functional subtypes of IKZF1 and those with non-functional subtypes of IKZF1 in the SR group (P>0.05). However, the predicted 5-year RFS rate of the children with functional subtypes of IKZF1 was significantly higher than that of those with non-functional subtypes of IKZF1 in the IR group and the HR group (P<0.05).

CONCLUSIONSB-ALL children with non-functional subtypes of IKZF1 have a high recurrence rate, and the recurrence rate will be even higher in B-ALL children with non-functional subtypes of IKZF1 and MRD ≥10 on day 33 of chemotherapy.

Antineoplastic Combined Chemotherapy Protocols ; Child ; Child, Preschool ; Female ; Genotype ; Humans ; Ikaros Transcription Factor ; genetics ; Male ; Neoplasm, Residual ; genetics ; mortality ; therapy ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; mortality ; therapy ; Prognosis ; Recurrence ; Remission Induction ; Survival

OBJECTIVE Regulating P2x7R- NLRP3 inflammasome activation might be a potentialtherapeutic strategy to treat alcoholic hepatosteatosis. We investigated whether this process would be modulated by gentiopicroside (GPS), which is attributed to the bitterness of gentian root extract. METHODSAn in vivo model was established by intragastrically treating mice with ethanol, and an in vitro modelwas created by treating HepG2 cells with ethanol or treating RAW 264.7 macrophages and murinebone marrow- derived macrophages (BMDMs) with lipopolysaccharides (LPS) plus adenosine triphos-phate (ATP). RESULTS In alcoholic hepatosteatotic mice model, GPS decreased serum aminotrans-ferase and triglyceride accumulation. GPS regulated sterol regulatory element-binding protein-1 (Srebp1),peroxisome proliferators- actived receptors α (PPARα) and acetyl CoA carboxylase (ACC) expressionvia elevating liver kinase B1 (LKB1)/AMP-activated Kinase (AMPK). Suppression of nucleotide-bindingoligomerization domain-like receptor protein 3 (NLRP3), caspase-1 and expression by GPS resulted inthe inhibition of interleukin-1β (IL-1β) production. In ethanol-exposed HepG2 cells, GPS reduced lipo-genesis and promoted lipid oxidation via P2x7R- NLRP3 inflammasome activation. P2x7R silencingenhanced AMPK activity, and reduced Srebp1 expression in ethanol-treated hepatocytes. GPS down-regulated P2x7R-mediated inflammatory response to extracellular ATP in LPS-primed RAW 264.7 macro-phages and BMDMs. Additionally, P2x7R deficiency attenuated IL- 1β cleavage in RAW 264.7 macro-phages, and GPS further suppressed IL-1β cleavage. CONCLUSION Activation of LKB1/AMPK signalingby GPS might be mediated by P2x7R-NLRP3 inflammasome, suggesting a therapeutic utility of P2x7Rblockade in alcoholic hepatosteatosis treatment.

OBJECTIVE The current study was designed to investigate the anti-steatosis effect of Pleurotus citrinopileatus extract (PC) and the underlying mechanism in vivo and in vitro. METHODS Acute and chronic alcoholic hepatosteatosis murine models and ethanol-treated HepG2 cells were applied. RESULTS In vitro,the anti-steatosis effect of PC was further confirmed via Nile red staining in HepG2 cells treated with ethanol.Both of acute and chronic alcohol-induced mice hepatosteatosis model,PC decreased serum aminotransferase and triglyceride accumulation. Upregulated sterol-regulatory element binding protein1(Srebp1),purinergic ligand-gated ion channel 7receptor(P2X7R)and downregulated sirtuin1 (SIRT1), adenosine 5′-monophosphate (AMP)-activated protein kinase α (AMPKα) caused by acute and chronic alcohol intake were modulated by PC.In ethanol-exposed HepG2 cells,PC reduced lipid accumulation in a concentration-dependent manner and exhibited superior ability in controlling lipid accumulation compared with metformin. CONCLUSION PC could abolish hepatic lipid accumulation through regulating SIRT1-AMPKα signaling in acute and chronic alcohol-induced hepatic steatosis.

OBJECTIVE Dihydroquercetin(TAX)is the most abundant dihydroflavone found in on-ions,milk thistle and Douglas fir bark.We investigated whether TAX could inhibit the lipid accumulation in alcoholic liver steatosis in vivo and in vitro.METHODS An in vivo model was established by intragas-trically treating mice with ethanol,and an in vitro model was created by treating HepG2 cells with etha-nol.RESULTS TAX regulated Sterol Regulatory Element-binding Protein-1(SREBP1)and Acetyl CoA Carboxylase (ACC) expression via elevating Liver Kinase B1 (LKB1)/ AMP-activated Kinase (AMPK) phosphorylation. Also, TAX upregulated SIRT1 expression, which suppressed by ethanal intake. Suppression of Purinergic 2X7 receptor (P2x7R), nucleotide-binding oligomerization domain-like re-ceptor protein 3(NLRP3)and Cysteine protease-1(caspase-1)cleavage by TAX resulted in the inhibi-tion of Interleukin-1β(IL-1β) production and release. Additionally, TAX reduced lipogenesis and pro-moted lipid oxidation via the regulation of AMPK and ACC in ethanol-treated steatotic HepG2 cell.TAX downregulated IL-1β cleavage response to Lipopolysaccharides (LPS) plus adenosine triphosphate(ATP) stimulation in HepG2 cells. P2x7R deficiency attenuated lipid accumulation with increasing AMPK activity and decreasing SREBP1 expression in ethanol-treated HepG2 cells.CONCLUSION Our data showed that TAX exhibited the inhibitory properties on lipogenesis and hepatoprotective ca-pacity,indicating that TAX has therapeutic potential for preventing alcoholic liver steatosis.

To determine the process parameters of optimal water-extraction and ethanol precipitation method for Xuanbi'antong (XBF) extract, which is a clinically experience formula for coronary disease. Orthogonal test L9(34) was conducted for the study of XBF water-extraction and ethanol precipitation process. Extractum, salvianolic acid B, rhizoma coptidis alkaloid, paeoniflorin, puerarin, ginsenoside Rb₁, ginsenosides and echinacoside were selected as marker components and multi-index comprehensive weighted score was used to select and verify optimal water-extraction and ethanol precipitation process. The optimal extraction process was as follows： XBF was added with 10 times distilled water, decocted for half an hour for 3 times. The best ethanol-precipitation process was established where the ethanol was added up to 70% and precipitated for 24 hours in 1.12 extract density (20 ℃). The optimized water-extraction and ethanol precipitation method is stable and reliable, and can provide reference for further development and utilization of the formula.

This study was purposed to investigate the effects of N-Arachidonoylethanolamine (ANA) on the quality of platelets (Plt) stored in Plt M-sol preservative solution at 22 ± 2°C. Samples taken from collecting apheresis Plt by the Amicus instrument and splited into two equal parts were stored in Plt M-sol preservative solution on a shaker at 22 ± 2°C. Different working concentrations of ANA (from 0.1 to 50 µmol/L) were then added into one part of stored Plt as the experimental group, the other without ANA was used as the control group. The viability of Plts stored at 22 ± 2°C for 7 days was evaluated by MTT colorimetric assay. The most effective concentration of ANA was selected and added to the subsequent experimental group. Plt count (BPC), mean Plt volume (MPV), Plt distribution width (PDW), phosphatidyl serine (PS) and soluble P-selectin were detected on the 1(st), 5(th), 7(th), 9(th) and 11(th) day of storage. The results showed that the most effective working concentration of ANA was 0.5 µmol/L, which showed significant increasing Plt viability (91.23 ± 5.44%) compared to the control group (62.54 ± 4.79%). Thus, ANA concentration at 0.5 µmol/L was choose to perform subsequent experiments. During 11 days of storage, the BPC, MPV and PDW were not changed significantly between the experimental group and control group, although there was decreasing trend in the BPC and increasing trends in MPV and PDW in the two groups. The rate of Plt PS positive was enhanced during the storage period: the rate of PS positive in experimental group increased from 7.69 ± 1.82% to 10.74 ± 1.78% while it in control group increased from 11.21 ± 2.03% to 15.37 ± 1.95%, with significant differences between the two groups (P < 0.05) on the 9(th) and 11(th) day of storage, respectively. Soluble P-selectin contents in experimental group on the 9(th) and 11(th) day of storage were 30.19 ± 2.03 ng/ml and 34.52 ± 2.64 ng/mL, respectively, while those in control group were 39.18 ± 2.66 ng/ml and 43.23 ± 2.58 ng/ml, respectively, with significant differences between the two groups (P < 0.05). It is concluded that the extended storage of Plt in M-sol treated with low concentration ANA can potentially alleviate Plt storage lesions.
Adult ; Blood Platelets ; drug effects ; Blood Preservation ; Endocannabinoids ; pharmacology ; Female ; Humans ; Male

OBJECTIVETo investigate the change in dendritic cells (DCs) in children with chronic immune thrombocytopenia (cITP) and the effect of glucocorticoid on DCs in children with cITP.

METHODSFifteen children with cITP and 20 healthy controls were included in the study. Flow cytometry was used to measure the DC subsets count in the 15 children with cITP before and after glucocorticoid treatment as well as the corresponding values in the 20 healthy controls. The DCs derived from peripheral blood monocytes in children with cITP were cultured in vitro and collected, and their immunophenotypes were determined by flow cytometry.

RESULTSBefore glucocorticoid treatment, the children with cITP showed no notable change in the absolute count of myeloid DCs (mDCs) but showed decreased absolute count of plasmacytoid DCs (pDCs) and increased mDC/pDC ratio compared with the healthy controls (P<0.05). After glucocorticoid treatment, the children with cITP demonstrated increased absolute count of pDCs and decreased absolute count of mDCs and mDC/pDC ratio compared with before treatment (P<0.05). Before glucocorticoid treatment, the children with cITP had significantly higher positive rates of HLA-DR, CD80, CD83 and CD86 on peripheral blood DCs than the healthy controls (P<0.01). All the positive rates were significantly decreased after glucocorticoid treatment (P<0.01), so that there was no significant difference from the healthy controls (P>0.05).

CONCLUSIONSDisproportion and functional disturbance of DC subsets is associated with the pathogenesis of cITP in children. Glucocorticoid can strengthen the immunosuppression of DCs in children with cITP, which may contribute to the effectiveness of glucocorticoid as a treatment.

Adolescent ; Child ; Child, Preschool ; Chronic Disease ; Dendritic Cells ; drug effects ; immunology ; Female ; Glucocorticoids ; pharmacology ; Humans ; Immunophenotyping ; Male ; Thrombocytopenia ; drug therapy ; immunology