The complex roles of m^(6)A modifications in neural stem cell proliferation, differentiation, and self-renewal and implications for memory and neurodegenerative diseases

N6-methyladenosine(m^(6)A), the most prevalent and conserved RNA modification in eukaryotic cells, profoundly influences virtually all aspects of mRNA metabolism. mRNA plays crucial roles in neural stem cell genesis and neural regeneration, where it is highly concentrated and actively involved in these processes. Changes in m^(6)A modification levels and the expression levels of related enzymatic proteins can lead to neurological dysfunction and contribute to the development of neurological diseases. Furthermore, the proliferation and differentiation of neural stem cells, as well as nerve regeneration, are intimately linked to memory function and neurodegenerative diseases. This paper presents a comprehensive review of the roles of m^(6)A in neural stem cell proliferation, differentiation, and self-renewal, as well as its implications in memory and neurodegenerative diseases. m^(6)A has demonstrated divergent effects on the proliferation and differentiation of neural stem cells. These observed contradictions may arise from the time-specific nature of m^(6)A and its differential impact on neural stem cells across various stages of development. Similarly, the diverse effects of m^(6)A on distinct types of memory could be attributed to the involvement of specific brain regions in memory formation and recall. Inconsistencies in m^(6)A levels across different models of neurodegenerative disease, particularly Alzheimer's disease and Parkinson's disease, suggest that these disparities are linked to variations in the affected brain regions. Notably, the opposing changes in m^(6)A levels observed in Parkinson's disease models exposed to manganese compared to normal Parkinson's disease models further underscore the complexity of m^(6)A's role in neurodegenerative processes. The roles of m^(6)A in neural stem cell proliferation, differentiation, and self-renewal, and its implications in memory and neurodegenerative diseases, appear contradictory. These inconsistencies may be attributed to the timespecific nature of m^(6)A and its varying effects on distinct brain regions and in different environments.

Silencing Bmi-1 enhances the senescence and decreases the metastasis of human gastric cancer cells

AIM:To evaluate the impact of Bmi-1 on cell senescence and metastasis of human gastric cancer cell line BGC823.METHODS:Two pairs of complementary small hairpin RNA(shRNA)oligonucleotides targeting the Bmi-1gene were designed,synthesized,annealed and cloned into the pRNAT-U6.2 vector.After DNA sequencing to verify the correct insertion of the shRNA sequences,the recombinant plasmids were transfected into BGC823 cells.The expression of Bmi-1 mRNA and protein was examined by reverse transcription-polymerase chain reaction(RT-PCR)and Western blotting.The effects of Bmi-1 knockdown on cell senescence and metastasis were determined by theβ-Gal activity assay and Boyden chamber assay,respectively.RESULTS:The double-stranded oligonucleotide fragments of Bmi-1 short interfering RNA(siRNA)cloned into pRNAT-U6.2 vector conformed to the inserted sequence.RT-PCR and Western blotting indicated that the expression levels of Bmi-1 gene mRNA and protein were markedly decreased in transfected BGC823 cells with pRNAT-U6.2-si1104 and pRNATU6.2-si1356,especially in transfected BGC823 cells with pRNAT-U6.2-si1104,compared with two control groups(empty vector and blank group).In particular,Bmi-1 protein expression was almost completely abolished in cells transfected with the recombinant vector harboring shRNA targeting the sequence GGAGGAGGTGAATGATAAA(nt1104-1122).Compared with untransfected cells and cells transfected with the empty vector,the mean percentage of senescent cells increased and the number of cells passing through the Matrigel decreased in cells transfected with the recombinant vectors.CONCLUSION:Silencing Bmi-1 by RNA interference can increase the senescent cell rate and effectively reduce the metastasis of gastric cancer cells.

Immunization of mice with concentrated liquor from male zooid of Antheraea pernyi

AIM: To study the effects of concentrated liquor from male zooid of Antheraea pernyi on immunological mice.METHODS: For each experiment, 40 mice were randomly divided into normal saline group (control group) and three tested groups that were administered different dosages of concentrated liquor from male zooid of A. pernyi and food for 15 d. The typical FSR and HC50 value, monocyte-phagocytic exponent Kand emendated monocyte-phagocytic exponent α were determined and calculated respectively.RESULTS: After 24 and 48 h, the FSR values of the three tested groups improved significantly in comparison to the control group by variance analysis. The HC50 values showed a significant difference between the high dosage group and the control group, as well as between the high dosage group and other two tested groups. The monocyte-phagocytic exponent Kand emendated exponent α showed rising tendencies, but no significant differences were found by variance analysis.CONCLUSION: The concentrated liquor from male zooid of A. pernyi can significantly enhance cellular and humoral immune function in mice, but has no distinct influence on the monocyte-phagocytic system in mice.

DETECTION OF PLATELET-DERIVED MICROPARTICLES USING FLOW CYTOMETRY AND ITS CLINICAL APPLICATION

Objective.To establish a flow cytometric internal standard method for counting platelet-derived microparti-cles(PMPs)and to study its clinical significance. Methods. PMPs suspension(platelet poor plasma,PPP) was extracted by gradual centrifugation. According to the size of PMPs,3 μm and 0.8μm latex beads were used as internal standards for the quantitation. PMPs were counted by adjusting flow cytometric discrimination and voltage of forward scatter and side scatter. Results. In 30 healthy donors,the average concentration of resting PMPs was(1.2×105±5.7×104 )/ml and that of activated PMPs was(1.6×106±9.1×105)/ml. Compared with healthy donors,PMPs mean value was significantly higher(P< 0.001)in 18 patients with coronary artery disease,12 with acute cerebral infraction and 23 with chronic renal failure[the average PMPs concentration,( 6.1×105±2.5×105 )/ml, ( 6.8×105±3.4×105)/ml and(5.9×105±3.1×105)/ml respectively]. However,no significant difference in PMPs concentration was observed in 25 patients with acute leukemia and severe thrombocytopenia during the aplastic phase after chemotherapy [1.3×105±6.1×104)/ml,(P >0.05)] .Conclusions. PMPs is a useful indicator in monitoring platelet activation,and plays an important role in thrombotic disease. By flow cytometric internal standard method,PMPs can be counted rapidly and accurately,which may be very helpful in interlaboratory comparative studies.

Elevated Plasma Tissue-type Plasminogen Activator (t-PA) and Soluble Throm-bomodulin in Patients Suffering From Severe Acute Respiratory Syndrome (SARS) as a Possible Index for Prognosis and Treatment Strategy

To detect the presence of endothelial injury in patients with severe acute respiratory syndrome （SARS） via enhanced levels of tissue-type plasminogen activator （t-PA） and soluble thrombomodulin （sTM）. Methods Case patients were from Xuanwu Hospital （Capital University of Medical Sciences, Beijing, China）, and all of them met clinical criteria for SARS. Healthy controls were some of the hospital employees. Endothelial injury bio-markers tPA and sTM were detected by commercial ELISA-methods. Results Classic plasma markers of endothelial injury, tPA and sTM significantly elevated in SARS patients in comparison to controls [t-PA： 1.48±0.16 nmol/L versus 0.25±0.03 nmol/L （P〈0.0001）, and sTM： 0.26±0.06 nmol/L versus 0.14±0.02 nmol/L （P〈0.05）]. The only patient who died had extremely high levels of these endothelial injury markers （t-PA： 2.77 nmol/L and sTM： 1.01 nmol/L）. The likelihood ratio analysis indicated the excellent discriminating power for SARS at the optimal cut-point of 0.49 nmol/L for tPA and 0.20 nmol/L for sTM, respectively. Significant numerical correlations were found among these endothelial injury markers in SARS patients. The numerical coefficient of correlation Pearson r between t-PA and sTM was 0.5867 （P〈0.05）. Conclusion Increased plasma concentrations of tPA and sTM in patients with SARS suggest the possibility of endothelial injury. SARS patients might need anticoagulant therapy or fibrinolytic therapy in order to reverse intraalveolar coagulation, microthrombi formation, alveolar and interstitial fibrin deposition. It may not only provide a useful treatment and prognostic index but also allow a further understanding of the pathological condition of the disease.

ANTITUMOR EFFECTS OF WICK OF SUN-FLOWER STEM IN MICE

A well-known Chinese traditional drug, decoction of wick of sun-flower stem, has been used to treat cancer and certain other diseases.1,2 Animal experiments in mice showed that transplantation of Sarcoma 180 (S180) and of Uterus cervical cancer 14 (U14) were signlflcanly inhibited by this decoction. The Inhibition rates of tumor weight were 33 -81 % and in about 6 - 20% of treated mice, complete tumor regression has been demonstrated. So tar no acute toxic side-effect was noted. This is in contrast to most of the antitumor chemltherapeutic drugs known to produce different degrees of toxlcity or induce Immunosuppression as a side effect. For this reason, we have undertaken the present study.

Application of Monoclonal Antibody in the Differential Diagnosis of Neuroblastoma and Leukemia in Children

Thirty-six cases of neuroblastoma and two hundredand twenty-nine cases with various kinds of leukemia inchildren were observed systematically. The applicationof monoclonal antibody in the differential diagnosis ofmetastasis of neuroblastoma cells in bone marrow and leu-kemia cells was studied and some precautions should betaken for a rapid and accurate diagnosis. The accordantrate between clinical diagnoses and the results of thisstudy was 91%.

LPS诱导的胸腺细胞凋亡对瘦素的抑制作用(英文)

Objective To study the signaling transduction mechanism of leptin, including phosphatidylinositol 3-kinase/AKT (PI3-K/AKT) and cPLA2 signal pathway. Methods MTT colorimetric assay and Annexin V-FITC/PI double staining for flow cytometry were used to demonstrate the inhibitory effect of leptin on lipopolysaccharide(LPS)-induced toxicity and apoptosis of thymocytes with a cPLA2 activity kit. RT-PCR was performed to show the suppressive effect of leptin on cPLA2 activity and cPLA2 mRNA level in proteins and genes. Effect of a specific inhibitor (LY294002) of PI3-K on apoptosis of thymocytes was detected by flow cytometry. Caspase3 mRNA was determined by RT-PCR. Results Leptin inhibited lipopolysaccharid(eLPS)-induced toxicity and apoptosis of thymocytes, decreased the cPLA2 activity and cPLA2 mRNA level in proteins and genes. Conclusion Leptin inhibits toxicity and apoptosis of thymocytes through the PI3-K/AKT and cPLA2 signal pathway.

Identification of Driver Genes in Primary Liver Cancer by Integrating NGS and TCGA Mutation Data

Background: This study is aimed towards an exploration of mutant genes in primary liver cancer (PLC) patients by using bioinformatics and data mining techniques. Methods: Peripheral blood or paraffin-embedded tissues from 8 patients with PLC were analyzed using a 551 cancer-related gene panel on an Illumina NextSeq500 Sequencer (Illumina). Meanwhile, the data of 396 PLC cases were downloaded from The Cancer Genome Atlas (TCGA) database. The common mutated genes were obtained after integrating the mutation information of the above two cohorts, followed by functional enrichment and protein-protein interaction (PPI) analyses. Three well-known databases, including Vogelstein’s list, the Network of Cancer Gene (NCG), and the Catalog of Somatic Mutations in Cancer (COSMIC) database were used to screen driver genes. Furthermore, the Chi-square and logistic analysis were performed to analyze the correlation between the driver genes and clinicopathological characteristics, and Kaplan-Meier (KM) method and multivariate Cox analysis were conducted to evaluate the overall survival outcome. Results: In total, 84 mutation genes were obtained after 8 PLC patients undergoing gene mutation detection with next-generation sequencing (NGS). The top 100 most mutate gene data from PLC patients in TCGA database were downloaded. After integrating the above two cohorts, 17 common mutated genes were identified. Next, 11 driver genes were screened out by analyzing the intersection of the 17 mutation genes and the genes in the three well-known databases. Among them, RB1, TP53, and KRAS gene mutations were connected with clinicopathological characteristics, while all the 11 gene mutations had no relationship with overall survival. Conclusion: This study investigated the mutant genes with significant clinical implications in PLC patients, which may improve the knowledge of gene mutations in PLC molecular pathogenesis.

A Clinical Study on the Effects and Mechanism of Xuebijing Injection (血必净注射液) in Severe Pneumonia Patients

Objective: To observe the effects of Xuebijing Injection (血必净注射液) in patients with severe pneumonia, and to explore the mechanism. Methods: Eighty cases of severe pneumonia are randomly assigned to the Xuebijing treatment (forty cases) and the control group (forty cases), with the same routine therapy provided in both groups. Clinical effective rates, inflammatory factors and organ function were observed in both groups. Results: The effective rate was higher in Xuebijing group than that of the control group (80.0% vs. 67.5%, P<0.05). As compared with the control group, the LDH, α1-AG, α1-AT levels and the peak body temperature decreased markedly with the Xuebijing treatment going, and the secretion of TNF-α, IL-6, IL-8 was suppressed in Xuebijing group; but no significant difference was found in leptin level. Conclusion: Xuebijing Injection may show a protective effect in patients with severe pneumonia. The mechanism is possibly with the decreased secretion of TNF-α, IL-6, and IL-8.

Molecular mechanisms of diabetic coronary dysfunction due to large conductance Ca2＋-activated K＋ channel impairment

Background Diabetes mellitus is associated with coronary dysfunction, contributing to a 2- to 4-fold increase in the risk of coronary heart diseases. The mechanisms by which diabetes induces vasculopathy involve endothelial-dependent and -independent vascular dysfunction in both type 1 and type 2 diabetes mellitus. The purpose of this study is to determine the role of vascular large conductance Ca2＋-activated K＋ （BK） channel activities in coronary dysfunction in streptozotocin-induced diabetic rats. Methods Using videomicroscopy, immunoblotting, fluorescent assay and patch clamp techniques, we investigated the coronary BK channel activities and BK channel-mediated coronary vasoreactivity in streptozotocin-induced diabetic rats. Results BK currents （defined as the iberiotoxin-sensitive K＋ component） contribute （65＋4）% of the total K＋currents in freshly isolated coronary smooth muscle cells and 〉50% of the contraction of the inner diameter of coronary arteries from normal rats. However, BK current density is remarkably reduced in coronary smooth muscle cells of streptozotocin-induced diabetic rats, leading to an increase in coronary artery tension. BK channel activity in response to free Ca2＋ iS impaired in diabetic rats. Moreover, cytoplasmic application of DHS-1 （a specific BK channel i~ subunit activator） robustly enhanced the open probability of BK channels in coronary smooth muscle cells of normal rats. In diabetic rats, the DHS-1 effect was diminished in the presence of 200 nmol/L Ca2＋ and was significantly attenuated in the presence of high free calcium concentration, i.e., 1 μmol/L Ca2＋. Immunoblotting experiments confirmed that there was a 2-fold decrease in BK-β1 protein expression in diabetic vessels, without alterinq the BK channel a-subunit expression.Although the cytosolic Ca2＋ concentration of coronary arterial smooth muscle cells was increased from （103±23） nmol/L （n=5） of control rats to （193±22） nmol/L （n=6, P 〈0.05） of STZ-induced diabetic rats, reduced BK-β1 expression made these channels less sensitive to intracellular Ca2＋, which in turn led to enhanced smooth muscle contraction. Conclusions Our results indicated that BK channels are the key determinant of coronary arterial tone. Impaired BK channel function in diabetes mellitus is associated with down-regulation of BK-β1 expression and reduction of the β1-mediated BK channel activation in diabetic vessels.

Identification of binding epitope of a monoclonal antibody (Z12) against human TNF-α using computer modeling and deletion mutant technique

The genes of the heavy and light chain variable region (VH, VL) of Z12 antibody against hTNF-a were cloned, and according to the translated sequence of amino acids, the spa-tial structures of VH and VL domains were modeled by using homology-based modeling method, followed by constructing the whole three-dimensional structure of Fv fragment. The complex model of Fv interacting with hTNF-a was gained with computer-guided molecular docking method, based on which, it was predicted that the epitope recognized by Z12 was from 141 to 146 of hTNF-a. hTNF-a molecule was divided into two fragments of N-terminal region from 1 to 91 and C-terminal region from 92 to 157 with prokaryotic expression. The measured results suggested that the antigenic epitope recognized by Z12 antibody was located in the C-terminal region 92-157 of hTNF-a, proving the predicted result reliable preliminarily. Further experimental results showed that after hTNF- 141-146 residues were deleted, Z12 antibody almost lost the ability to recognize the mutant, suggesting that the amino acid residues from 141 to 146 of hTNF-a were specially recognized by Z12 antibody.

Composition of Ophiopogon Polysaccharide,Notoginseng Total Saponins and Rhizoma Coptid is Alkaloids Inhibits Myocardial Apoptosis in Diabetic Atherosclerosis Rabbits

Objective: To investigate the effects of Composition of Ophiopogon polysaccharide, Notoginseng total saponins and Rhizoma Coptidis alkaloids(CONR) on myocardial apoptosis of diabetic atherosclerosis(DA) rabbits Methods: Sixty male New Zealand white rabbits were randomly divided into 6 groups [control group, model group, CONR high-dose group(450 mg/kg), CONR medium-dose group(150 mg/kg), CONR low-dose group(50 mg/kg), and simvastatin group] by using a completely random method, 10 in each group. DA model was established by intravenously injected alloxan combined with high-fat diet and abdominal aortic balloon injury. After mediation for 10 weeks, fasting blood glucose(FBG), glycosylated hemoglobin(GHB), glycosylated serum protein(GSP), fructoseamine(FRA), aldose reductase(AR), advanced glycation end products(AGEs) in serum were measured by enzyme linked immunosorbent assay(ELISA) method;the expression of receptor of AGEs(RAGE) in myocardial tissue were observed by immunohistochemical method;and p-Jun N-terminal kinase(p-JNK), caspase-3, B-cell lymphoma-2(bcl-2) protein expression in myocardial tissue were measured by Western blotting. The myocardial apoptosis was detected by Td T-mediated d UTPnick-end labeling(TUNEL) method, and apoptosis index(AI) was calculated. Results: Compared with the control group, serum FBG, GHB, GSP, FRA, AR, AGEs and the expression of myocardium RAGE, p-JNK, caspase-3 proteins, as well as apoptosis index(AI) were significantly increased and bcl-2 protein was significantly decreased in the model group(P<0.01). Compared with the model group, the levels of serum FBG, GHB, GSP, FRA and AR showed a significant decline in CONR high-and medium-dose groups(P<0.01). FBG and GHB showed a significant decline in CONR low-dose group(P<0.01). Compared with the model group, the expression of serum AGEs and myocardium RAGE, p-JNK and caspase-3 protein as well as AI were significantly decreased and bcl-2 protein was significantly up-regulated in all treatment groups(P<0.01);high-dose CONR had the most significant effect on abovementioned indices compared with other treatment groups(P<0.01). Middle-dose CONR had better effect on serum AGEs compared with the low-dose group(P<0.01);middle-dose CONR and simvastatin groups had better effect on the expression of caspase-3, bcl-2 protein, myocardium apoptosis compared with the CONR low-dose group(P<0.01). Conclusion: CONR may effectively inhibit myocardial apoptosis on DA rabbits by intervening AGEs-RAGE and JNK, caspase-3, and bcl-2 protein expressions.

Analysis of low-density lipoprotein receptor gene mutations in a Chinese patient with clinically homozygous familial hypercholesterolemia

Objective To screen the point mutation of the low-density lipoprotein receptor (LDL-R) gene in Chinese familial hypercholesterolemia (FH) patients,characterize the relationship between the genotype and the phenotype and discuss the molecular pathological mechanism of FH. Methods A patient with clinical phenotype of homozygous FH and her parents were investigated for mutations in the promoter and all eighteen exons of the LDL-R gene. Screening was carried out using Touch-down PCR and direct DNA sequencing; multiple alignment analysis by DNASIS 2.5 was used to find base alteration,and the LDL-R gene mutation database was searched to identify the alteration. In addition,the apolipoprotein B gene (apo B) was screened for known mutations (R3500Q) that cause familial defective apo B 100 (FDB) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).Results Two new heterozygous mutations in exons 4 and 9 of the LDL-R gene were identified in the proband (C122Y and T383I) as well as her parents. Both of the mutations have not been published in the LDL-R gene mutation database. No mutation of apo B 100 (R3500Q) was observed. Conclusion Two new mutations (C112Y and T383I) were found in the LDL-R gene,which may result in FH and may be particularly pathogenetic genotypes in Chinese people.

针刺相对穴对穴区肥大细胞及其释放的组织胺和缓激肽的影响（英文）

目的:观察针刺大鼠相对穴阳陵泉与阴陵泉后穴区局部肥大细胞数量、脱颗粒情况及血清中组织胺、缓激肽的含量变化,探索"相对穴"协同增效的作用机制。方法:将40只Wistar大鼠采用随机数字表法随机分成空白对照组(K组)、针刺阳陵泉组(A组)、针刺阴陵泉组(B组)、针刺阳陵泉与阴陵泉组(AB组)及针刺阳陵泉与阴陵泉旁开组(CD组),每组8只。A组针刺阳陵泉;B组针刺阴陵泉;AB组针刺阳陵泉和阴陵泉;CD组分别采用阳陵泉和阴陵泉外侧旁开3 mm处作为旁开对照点,进行针刺。均选取双侧穴位。K组实验开始后采用与其余4组相同的方法固定,不给予针刺刺激。选用直径0.35 mm,长40 mm毫针,针刺得气后针柄接G6805-Ⅱ型电针治疗仪,选用疏密波,频率2 Hz/100 Hz,电流强度1 mA,以针柄轻微颤动且大鼠能保持安静为宜。每次电针持续刺激20 min。在开始后的第1、3、5、7天进行实验干预,共4次。各组大鼠于第7天干预完成2 h后取材。采用酶联免疫吸附法(ELISA)检测血清中组织胺和缓激肽的含量变化;穴区局部组织做冰冻切片,甲苯胺蓝染色,观察穴区肥大细胞(MCs)的变化。结果:与K组相比,A组、B组、AB组及CD组的MC数量及脱颗粒率均明显升高(均P<0.05),其中AB组的MC数量及脱颗粒率明显高于A组、B组和CD组(均P<0.05);组织胺含量由高到低的顺序为AB组>B组>A组>CD组>K组,且差异有统计学意义(均P<0.05);AB组、A组和B组的缓激肽含量明显高于K组和CD组(均P<0.05),A组和B组缓激肽含量差异,CD组与K组缓激肽含量差异均无统计学意义(均P>0.05)。结论:针刺"相对穴"阳陵泉与阴陵泉后穴区局部MC数量及脱颗粒率显著增高,血清中组织胺和缓激肽的含量升高,可能是"相对穴"配伍协同增效的机制之一。

Prediction on the binding domain between human interleukin-6 and its receptor

Based on the spatial conformations of human interleukin-6 (hlL-6) derived from nuclear magnetic resonance analysis and human interleukin-6 receptor (hlL-6R) modeled with homology modeling method using human growth hormone receptor as template, the interaction between hlL-6 and its receptor (hIL-6R) is studied with docking program according to the surface electrostatic potential analysis and spatial conformation complement. The stable region structure composed of hlL-6 and hlL-6R is obtained on the basis of molecular mechanism optimization and molecular dynamics simulation. The binding domain between hIL-6 and hIL-6R is predicted theoretically. Furthermore, the especial binding sites that influence the interaction between hlL-6 and hlL-6R are confirmed. The results lay a theoretical foundation for confirming the active regions of hlL-6 and designing novel antagonist with computer-guided techniques.