The advancement of tissue clearing technology has significantly propelled neuroscience research. Nevertheless, the fluorescent proteins used in traditional transgenic mouse strains were not specifically optimized for tissue clearing procedures, resulting in a substantial decrease in fluorescent intensity after clearing. In this study, we developed the Ci1 reporter mouse strain (where Ci stands for the Chinese Institute for Brain Research, CIBR) based on the bright red fluorescent protein mScarlet. The Ci1 reporter exhibits no fluorescence leakage in various organs or tissue types and can be readily crossed with multiple tissue-specific Cre lines. Compared to the Ai14 mouse strain, the Ci1 reporter strain demonstrates lower non-specific leakage, stronger fluorescence intensity in different tissues, and better preservation of fluorescence following tissue clearing treatment. The creation of the Ci1 reporter provides a more effective tool for both neuroscience and other biomedical research applications.
Animals ; Luminescent Proteins/metabolism* ; Mice, Transgenic ; Mice ; Red Fluorescent Protein ; Brain/metabolism* ; Genes, Reporter ; Fluorescence

【Objective】 To analyze the efficacy of ABO-matched platelet transfusions and ABO-mismatched platelet transfusions in patients with hematonosis and to explore the effect of circulating immune complexes (CIC) on the efficacy. 【Methods】 A total of 1 510 platelet transfusions involving 757 patients in our hospital from January 2013 to June 2018 were retrospectively analyzed. The patients were divided into ABO-matched group and ABO-mismatched group. The 12-hour percent platelet recovery (PPR) was used to evaluate the effect of platelet transfusion between the groups. TEG was used to evaluate the efficacy of the transfusions, and CIC value was measured before and after platelet transfusion. The effect of A-B/CIC (or AB-O/CIC) on platelet function was tested. 【Results】 1)The results showed that platelet transfusion was effective(PPR>30%) in both ABO-matched group[PPR=(66.5±52.8)%] and ABO-mismatched group[PPR=(47.7%±51.6)%], and there was no increase in the report of hemolytic transfusion reaction of ABO-mismatched group. The efficacy of ABO-matched platelet transfusions was significantly better than that of ABO-mismatched group(P < 0.01). If a patient had received multiple transfusions of ABO-mismatched platelet, there were no significance between ABO-matched group[PPR=(49.8%±51.8)%] and ABO-mismatched group[PPR=(47.7%±51.6)%], P>0.05. 2) In the experiment of simulating platelet transfusion in patients, no difference in MA value of TEG was noticed between ABO-mismatched groups and ABO-matched groups (all P>0.05). 3) There was no difference in CIC value before and after platelet transfusions (P>0.05) in the ABO-matched group, while CIC value decreased significantly in all ABO-mismatched groups (all P < 0.05). 4) The MA values (mm)of AB, A and O blood group platelets mixed with A-B/CIC and AB-O/CIC were 36.1 vs 31.1, 37.8 vs 35.0 and 43.1 vs 45.7, with the MA value (mm) in control group at 49.2 vs 49.5, respectively. 【Conclusion】 Platelet transfusion was effective in both ABO-matched group and ABO-mismatched group, and the efficacy of ABO-matched group was significantly better compared with the ABO-mismatched group. There was no increase in the safety risk of ABO-mismatched platelet transfusion with major mismatches/minor matches. CIC can inhibit the function of platelets and combine more with ABO-matched platelets than with ABO-mismatched platelets, therefore, CCI is an important influencing factor on the efficacy of platelet transfusions.

Background:Circular RNAs(circRNAs)are covalently closed single-stranded RNAs with multiple biological functions.CircRNA.0007127 is derived from the carbon catabolite repression 4-negative on TATA-less(CCR4-NOT)complex subunit 2(CNOT2),which was found to regulate tumor cell apoptosis through caspase pathway.Methods:Potential circRNA.0007127 target microRNAs(miRNAs)were analyzed by miRanda,TargetScan,and RNAhybrid software,and the miRNAs with binding sites for apoptosis-related genes were screened.The roles of circRNA.0007127 and its downstream target,microRNA(miR)-513a-5p,were validated by quantitative real-time polymerase chain reaction(qPCR),flow cytometry,mitochondrial membrane potential,immunofluorescence,western blot,and caspase-8(CASP8)protein activity in vitro in H2O2-induced K-562 cells.The circRNA.0007127-miR-513a-5p and CASP8-miR-513a-5p interactions were verified by luciferase reporter assays.Results:Silencing circRNA.0007127 decreased cell apoptosis by inhibiting CASP8 pathway activation in K-562 cells.Compared with the control group,the expression of CASP8 was reduced by 50％and the 43-kD fragment of CASP8 protein was significantly reduced(P≤0.05).The luciferase reporting assay showed that circRNA.0007127 combined with miR-513a-5p or CASP8,with extremely significant differences(P≤0.001).The overexpression of miR-513a-5p inhibited the gene expression level of CASP8 in a human myeloid leukemia cell model(75％change)and the level of a 43-kD fragment of CASP8 protein(P≤0.01).The rescue experiment showed that cotransfection with circRNA.0007127 small-interfering RNA(siRNA)and the miR-513a-5p inhibitor increased CASP8 gene expression and the apoptosis rate,suggesting that the miR-513a-5p inhibitor is a circRNA.0007127 siRNA antagonist.Conclusions:CircRNA.0007127 regulates K-562 cell apoptosis through the miR-513a-5p/CASP8 axis,which can serve as a novel powerful molecular target for K-562 cells.

Background: Blackened intestines in slaughtered pigs have been commonly observed in China in recent years. However, no cause has been reported. Objectives: We attempted to determine whether the blackening of the pig intestine was related to an excess of copper (Cu) in their feed. Methods: In this study, we observed and collected porcine intestines in small- and large-scale pig slaughterhouses in Shandong province from May to October 2018. Twelve types of metal ions were detected in the black intestinal samples. Results: The Cu level in the intestine samples was mostly higher than the Chinese national limit for food. Further study showed that Cu supplementation in most commercial porcine feed also exceeded the national standard. An animal model (mouse) that could mimic the intestinal blackening in pigs was established. Compared to control mice, Cu accumulated in the liver and intestines of mice fed an excessive Cu level, confirming the excessive Cu in the feed may be considered the major cause of blackened porcine intestines. Microscopic examination revealed that black intestines had many particles containing Cu in the lamina propria of the intestinal mucosa, and the intestinal mucosal epithelial cells showed degeneration and necrosis. Conclusions In conclusion, overuse of Cu in animal feed can lead to animal poisoning and Cu accumulation in animal products. Such overuse not only harms the health of livestock but can also affect public health.

Background: Blackened intestines in slaughtered pigs have been commonly observed in China in recent years. However, no cause has been reported. Objectives: We attempted to determine whether the blackening of the pig intestine was related to an excess of copper (Cu) in their feed. Methods: In this study, we observed and collected porcine intestines in small- and large-scale pig slaughterhouses in Shandong province from May to October 2018. Twelve types of metal ions were detected in the black intestinal samples. Results: The Cu level in the intestine samples was mostly higher than the Chinese national limit for food. Further study showed that Cu supplementation in most commercial porcine feed also exceeded the national standard. An animal model (mouse) that could mimic the intestinal blackening in pigs was established. Compared to control mice, Cu accumulated in the liver and intestines of mice fed an excessive Cu level, confirming the excessive Cu in the feed may be considered the major cause of blackened porcine intestines. Microscopic examination revealed that black intestines had many particles containing Cu in the lamina propria of the intestinal mucosa, and the intestinal mucosal epithelial cells showed degeneration and necrosis. Conclusions In conclusion, overuse of Cu in animal feed can lead to animal poisoning and Cu accumulation in animal products. Such overuse not only harms the health of livestock but can also affect public health.

Objective To investigate the correlations of serum cystatin C level with severity of stroke and short-term outcome in patients with acute ischemic stroke.Methods Patients with first-ever acute ischemic stroke aged ≥50 years who did not receive thrombolysis and took a visit within 3 d after onset were selected prospectively.The serum cystatin C level was detected within 24 h after admission and various clinical data were collected.The National Institutes of Health Stroke Scale (NIHSS) was used to assess the neurological deficits on the day of admission.The NIHSS score ＜8 was defined as mild stroke and ≥8 was defined as moderate to severe stroke.The modified Rankin Scale (mRS) was used to evaluate the short-term outcome at discharge or 14 d after onset,0-2 was defined as good outcome and ＞2 was defined as poor outcome.Results A total of 188 patients were enrolled,including 93 (49.5％) females and 95 (50.5％) males,their mean age was 65.4 ±9.2 years old (range 50-87).There were 120 patients with mild stroke (63.8％),68 with moderate to severe stroke (36.2％);106 patients (56.4％) had good outcome and 82 (43.6％) had poor outcome.Univariate analysis showed that serum cystatin C level in the moderate to severe stroke group was significantly higher than that in the mild stroke group (1.36 ± 0.29 mg/L vs.1.21 ±0.23 mg/L;t =3.902,P ＜ 0.001),the serum cystatin C level in the poor outcome group was significantly higher than that in the good outcome group (1.38 ± 0.25 mg/L vs.1.22 ± 0.25 mg/L;t =4.101,P =0.001).Multivariate logistic regression analysis showed that the serum cystatin C level was an independent risk factor for stroke severity (odds ratio 12.182,95％ confidence interval 11.163-13.202;P ＜ 0.001) and short-term poor outcome (odds ratio 9.025,95 ％ confidence interval 8.202-9.848;P ＜ 0.001).Conclusion The serum cystatin C level is significantly correlated with the severity of stroke and the short-term outcome in patients with acute ischemic stroke.

Objective To investigate the relationship betw een the serum bilirubin level and the severity of disease and short-term outcome in patient w ith acute ischemic stroke. Methods A total of 120 consecutive inpatients w ith acute ischemic stroke w ere enroled and 105 healthy subjects at the same time w ere used as a control group. The biochemical indicators, such as serum total bilirubin, direct bilirubin, indirect bilirubin, blood lipid, and blood glucose w ere measured w ithin 24 h after admission. The National Institutes of Health Stroke Scale ( NIHSS ) w as used to assess the neurological deficits on the day of admission. The NIHSS score <8 w as defined as mild stroke and ≥8 w as defined as moderate to severe stroke. At discharge or 14 d after onset, the modified Rankin Scale (mRS) w as used to evaluate the clinical outcomes, 0-2 w as defined as good outcome and > 2 w as defined as poor outcome. The levels of serum total bilirubin, direct bilirubin, and indirect bilirubin w ere measured again. Results The levels of serum total bilirubin, direct bilirubin, and indirect bilirubin in the moderate to severe stroke group w ere significantly higher than those in the mild stroke group ( P <0.01) and the control group ( P <0.01). Multivariate logistic regression analysis show ed that the increased levels of serum total bilirubin (odds ratio [OR] 1.855,95%confidence interval [CI] 1.390-2.475; P <0.01), indirect bilirubin ( OR 3.380, 95%CI 1.271-11.901; P <0.05), and direct bilirubin ( OR 3.51, 95%CI 1.062-11.473; P <0.01) had significantly independent correlation w ith baseline disease severity. Univariate analysis show ed that the increased serum total bilirubin level on admission w as associated w ith the short-term poor outcome ( P <0.05), but after adjustment for other confounding factors, there w as no statistical significance ( OR 2.411, 95%CI 0.803-7.243, P >0.05). Conclusions The serum bilirubin level show ed stress increase in patients w ith cerebral infarction in acute phase; and it w as significantly associated w ith the degree of neurological deficit, but it w as not associated w ith short-term outcome. It might be a defense response to the body for stroke events.

OBJECTIVETo investigate the influence of exposure to different concentrations of ethanol on neural progenitor cells and the differentiation of neurons and glial cells in zebrafish embryos.

METHODSZebrafish embryos were exposed to 1%, 2%, and 2.5% (V/V) ethanol at 5 hpf by adding ethanol to the egg water. In situ hybridization and real-time PCR were used to detect the changes in the mRNA expression profiles of the markers of different cells to examine the effects of alcohol on neural development.

RESULTSThe number of neural precursor cells, neurons and mature glial cells was significantly reduced in the zebrafish embryos following ethanol exposure, and this reduction became more prominent as the ethanol concentration increased. The expression of the early glial marker slc1a3a was down-regulated in the spinal cord but increased in the brain after exposure to increased ethanol concentrations. The expression of the mature glial markers was significantly lowered in response to exposure to increasing ethanol concentrations.

CONCLUSIONSEthanol can reduce neural precursor cells and inhibits neuronal and glial differentiation in zebrafish embryos.

Animals ; Brain ; Cell Differentiation ; drug effects ; Embryo, Nonmammalian ; drug effects ; Ethanol ; adverse effects ; Neural Stem Cells ; drug effects ; Neurogenesis ; drug effects ; Neuroglia ; drug effects ; Neurons ; drug effects ; Spinal Cord ; Zebrafish ; embryology

OBJECTIVETo investigate the effect of quercetin on apoptosis and feedback regulation of MDM2-p53 in multiform glioblastoma U87 cells in vitro.

METHODSU87 cells exposed to different concentrations of quercetin (50, 100, and 150 µmol/L) were examined with flow cytometry, RT-PCR and Western blotting for detecting the cell apoptosis, MDM2 mRNA expression, and p53 and caspase-3 expressions.

RESULTSQuercetin induced obvious apoptosis in U87 cells in a concentration-dependent manner, with apoptosis rates of (12.40∓0.70)% at Q0, (22.53∓0.72)% at Q50, (29.06∓0.81)% at Q100, and (31.5∓0.45)% at Q150. Quercetin significantly increased the expressions of MDM2 mRNA and active caspase-3 protein but decreased the expression of p53 in the cells.

CONCLUSIONQuercetin promotes the apoptosis of multiform glioblastoma U87 cells mediated by caspase-3 and influences the feedback balance of MDM2-p53.

Apoptosis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; drug effects ; Glioma ; metabolism ; pathology ; Humans ; Proto-Oncogene Proteins c-mdm2 ; metabolism ; Quercetin ; pharmacology ; Tumor Suppressor Protein p53 ; metabolism

Objective To investigate the effect of quercetin on apoptosis and feedback regulation of MDM2-p53 in multiform glioblastoma U87 cells in vitro. Methods U87 cells exposed to different concentrations of quercetin (50, 100, and 150μmol/L) were examined with flow cytometry, RT-PCR and Western blotting for detecting the cell apoptosis, MDM2 mRNA expression, and p53 and caspase-3 expressions. Results Quercetin induced obvious apoptosis in U87 cells in a concentration-dependent manner, with apoptosis rates of (12.40 ± 0.70)% at Q0, (22.53 ± 0.72)% at Q50, (29.06 ± 0.81)% at Q10 , and (31.5 ± 0.45)% at Q150. Quercetin significantly increased the expressions of MDM2 mRNA and active caspase-3 protein but decreased the expression of p53 in the cells. Conclusion Quercetin promotes the apoptosis of multiform glioblastoma U87 cells mediated by caspase-3 and influences the feedback balance of MDM2-p53.