N-methyladenosine reader YTHDF2-mediated long noncoding RNA FENDRR degradation promotes cell proliferation in endometrioid endometrial carcinoma.

Dysregulation of long noncoding RNA (LncRNA) FENDDR has been shown to be closely related to the progression of several cancers. However, its role and upstream regulatory mechanism in endometrioid endometrial carcinoma (EEC) remains unclear. This study was conducted using the cancerous tissues of EEC patients (n = 60), EEC cell lines, and a xenograft mouse model. The expression level of LncRNA FENDRR was decreased and the N-methyladenosine (m6A) methylation levels of LncRNA FENDRR was elevated in cancerous tissues of EEC patients. In vitro experiments demonstrated that YTH domain-containing 2 (YTHDF2), an m6A reader, recognized the abundance of m6A-modified LncRNA FENDRR in EEC cells and promoted its degradation. LncRNA FENDRR overexpression suppressed cell proliferation and facilitated cell apoptosis in the EEC cell line HEC-1B by reducing the protein level of SRY-related HMG box transcription factor 4 (SOX4). Interference of LncRNA FENDRR reversed the inhibitory effect of sh-YTHDF2 on cell proliferation and the promoting effect of sh-YTHDF2 on cell apoptosis in HEC-1B cells by silencing FENDRR. Finally, in vivo experiments confirmed that overexpression of LncRNA FENDRR retarded the growth of EEC cells. In conclusion, YTHDF2-mediated LncRNA FENDRR degradation promotes cell proliferation by elevating SOX4 expression in EEC.

[Stabilities of two kinds of Plasmodium antigen substrate slides under different storage temperatures and time].

OBJECTIVE: To observe and compare the inspection effects of antigen substrate slides of Plasmodium cynomolgi (P. c) and Plasmodium falciparum (P. f) on the malaria antibody titer under different storage temperatures and time. METHODS: The densities of Plasmodium of P. c and P. f antigen slides were counted through a microscope, and the average numbers of Plasmodium in each field of vision were calculated. The pooled serum of patients with tertian malaria and falciparum malaria were treated as antibody serum, and the dilutions were from 1:5 to 1:1 280. The two kinds of antibody slides were placed under the temperature of 4-6, 25-27, 33-35 degrees C, and two slides of each kind were selected at Day 3, 5, 7, 10 post-storage to detect the end point antibody titer of malaria by the indirect fluorescent antibody test. Meanwhile, the P. c and P. f antigen slides storing under -20 degrees C for 1 year and 2 years were placed under the aforementioned 3 temperature conditions for 3 d, and the changes of the antibody titers were compared. RESULTS: The densities of Plasmodium in blood cells of P. c and P. f antigen slides were 2.00 x 10(5)/microl and 1.89x 10(5)/microl, respectively, and the average numbers of Plasmodium in each field of vision were 157 +/- 13 and 142 +/- 9, respectively. The end point titers of antibody of P. c and P. f antigen slides placed under the temperatures of 4-6, 25-27, 33-35 degrees C were all on a downward trend after storing for 5 d. The average antibody titers of the two kinds of slides under temperature of 4-6 degrees C and above were 1:440 and 1:80, respectively, and there was a significantly statistic difference between them(t = 1.940, P < 0.05). When P. c and P. f antigen slides storing under -20 degrees C for 1 year were placed under 4-6 degrees C for 3 d, the end point antibody titers were both 1:640, while for those storing under -20 degrees C for 2 years, the end point antibody titers were 1:320 and 1:160, respectively, both the differences were statistically significant (t(P. c) =11.362, P(P. c) < 0.01; t(P. f) = 38.845, P(P. f) < 0.001). CONCLUSION: The end point antibody titers for malaria detection of P. c and P. f antigen slides decrease gradually with the raise of temperature and the prolonging of time for storage.

[Correlation of IL-4 and IL-13 gene polymorphisms with asthma and total serum IgE levels].

OBJECTIVE: To investigate the correlation between the single nucleotide polymorphisms (SNP) of +33C/T in the promoter region of IL-4 gene and +1923C/T in intron-3 region of IL-13 gene and the susceptibility of asthma, and to study the impact of these polymorphisms upon total serum IgE levels. METHODS: The 2 polymorphisms were determined by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP), in 150 asthmatic subjects (asthma group) and 160 healthy controls (healthy control group) of the Han nationality in Shandong province enrolled from December 2003 to June 2007. The total serum IgE levels were determined by ELISA. RESULTS: The genotype frequencies of CC, CT and TT in +33C/T sites of IL-4 gene were 43% (68/160), 35% (56/160), 22% (36/160) respectively in the controls, and 18% (27/150), 36% (54/150), 46% (69/150) respectively in the asthmatic subjects. The genotype frequencies of CC, CT and TT in +1923C/T sites of IL-13 gene were 41% (66/160), 43% (68/160), 16% (26/160) respectively in the controls, and 21% (31/150), 38% (57/150), 41% (61/150) respectively in the asthmatic subjects. The distribution of genotype in each sites between the 2 groups was significantly different (chi(2) = 27.821, 26.544 respectively, all P < 0.01). The CT and TT genotypes carried higher risks for asthma than CC genotypes (chi(2) = 21.870, 14.206 respectively, all P < 0.01). The total serum IgE levels of CC, CT and TT in +33C/T sites of IL-4 gene were (92 +/- 37), (122 +/- 45), (146 +/- 44) KU/L respectively in the controls, and (179 +/- 40), (294 +/- 51), (341 +/- 80) KU/L respectively in the asthmatic subjects. The total serum IgE levels of CC, CT and TT in +1923C/T sites of IL-13 gene were (85 +/- 31), (102 +/- 38), (144 +/- 49) KU/L respectively in the controls, and (186 +/- 65), (297 +/- 87), (363 +/- 140) KU/L respectively in the asthmatic subjects. In these 2 sites, the total serum IgE level of asthmatics was higher than that of the controls with the same genotype between the 2 groups (t = 4.653, 6.547, 7.754; and 4.673, 6.784, 8.157 respectively, all P < 0.01). The total serum IgE levels of CT, TT genotypes were higher than CC genotypes in the same group (t = 5.748, 6.253 respectively, all P < 0.01). CONCLUSIONS: There was a significant correlation between the polymorphisms of +33C/T sites of IL-4 and +1923C/T sites of IL-13 gene and susceptibility to asthma and increase of the total serum IgE. The IL-4 gene and IL-13 gene may be important candidate genes for asthma.

[Establishment of in vitro microtest for determining sensitivity of Plasmodium falciparum to pyronaridine].

OBJECTIVE: To establish an in vitro microtest for determining the sensitivity of Plasmodium falciparum to pyronaridine. METHODS: Pyronaridine-coated plate and culture medium which is easy to use in the field were prepared. P. falciparum parasites from in vitro continuous passage culture (FCC1/HN) were used for experimental tests in the laboratory. When they were proved stable and reliable through repeated determinations, field trials were made in Hainan and Yunnan Provinces during the malaria transmission season with blood samples from clinical falciparum malaria cases. A 4-week in vivo test was carried out as a control. RESULTS: The pyronaridine-coated plate and culture medium were proved to be stable. The effective period of pyronaridine-coated plate, the ampule sealed liquid culture medium and the bottled lyophilized culture medium, all stored at 4 degrees C was 6 months, 2 months and 2 years respectively. Through several years field determinations, the baseline data of pyronaridine-sensitivity of P. falciparum in the country were collected and the sensitivity of P. falciparum to pyronaridine was also revealed to have decreased gradually. The mean drug concentration for in vitro complete inhibition of schizont formation raised by 2-4 times although the clinical therapeutic efficacy of pyronaridine was still satisfactory at the present time. CONCLUSION: The developed in vitro microtest can be used for determination of the sensitivity of P. falciparum to pyronaridine, and it is more convenient and sensitive than the 4-week in vivo method.

[The point mutations in Pfcrt and Pfmdr1 genes in Plasmodium falciparum isolated from Hainan Province].

OBJECTIVE: To evaluate the point mutations in Pfcrt and Pfmdr1 genes in Plasmodium falciparum isolated from Hainan Province. METHODS: Nested polymerase chain reaction and restriction fragment length polymorphism were used to detect the point mutations at codon 76 of Pfcrt and at codon 86, 1246 of Pfmdr1 in P. falciparum isolates. Chloroquine resistance was measured by the in vitro microtest recommended by WHO. RESULTS: In 36 samples tested, 28 were successfully amplified for Pfcrt, 64.3% of them carried mutant allele at codon 76, 21.4% with wild allele K76 and 14.3% with mixed allele mutation. While for Pfmdr1, 3.4% isolates displayed the 86Y mutation, 89.7% with wild allele N86 and 6.9% with the mixed alleles in 29 isolates which were amplified successfully for N86Y. No point mutation in Pfmdr1 at codon 1246 was found in 13 isolates from the total 36 samples. By the in vitro test, 72.2% (26/36) showed resistance to chloroquine. The 76T and 86Y mutant alleles were present in both in vitro susceptible and resistant isolates. There was a significant difference between susceptible and resistant isolates carrying 76T mutant codon (P < 0.05), but no significant difference was found in Pfmdr1 (P < 0.05). CONCLUSION: There is a significant difference of the 76T prevalence in Pfcrt gene between the susceptible isolate and resistant one of P. falciparum to chloroquine in vitro. The Pfcrt 76T may be used as a predictive marker for chloroquine resistance surveillance.

[Fluctuation in the resistance of Plasmodium falciparum to chloroquine in China].

OBJECTIVE: To investigate whether chloroquine-resistance of Plasmodium falciparum had changed after stopping or reducing the use of chloroquine as an antimalarial in Hainan and Yunnan provinces. METHODS: WHO standard in vitro microtest and 4-week in vivo test were used, assays were carried out in different time after stopping or reducing the use of chloroquine. RESULTS: In vitro test in Hainan indicated that the rate of chloroquine-resistant P. falciparum was 97.9% in 1981, and dropped to 26.7% in 1997 (P<0.01). The mean concentration of chloroquine for complete inhibition of schizont formation was 10.46 +/- 7.14 pmol/microl blood in 1981, decreased to 1.63 +/- 1.47 pmol/microl blood in 1997 (P<0.01). The proportion of samples taken from malaria cases that required high concentration (>6.4 pmol/microl blood) of chloroquine for complete inhibition of schizont formation was 83.3% in 1981 and only 6.7% in 1997 (P<0.01). In the 4-week in vivo test, the rate of chloroquine-resistant P. falciparum decreased from 84.2% in 1981 to 18.4% in 1997 (P<0.01). RIII cases accounted for 53.1% of the total resistant cases in 1981, and for 14.3% in 1997 (P<0.01). In vitro test in Yunnan revealed that the rate of chloroquine-resistant P. falciparum, the mean concentration of chloroquine for complete inhibition of schizont formation and the proportion of samples taken from malaria cases that required >6.4 pmol/microl blood of chloroquine for complete inhibition of schizont formation were 97.4%, 17.2 +/- 12.6 pmol/microl blood and 58.9% in 1981 respectively, and dropped to 70.4% (P< 0.01), 4.0 +/- 3.3 pmol/microl blood (P<0.01) and 16.6% (P<0.01) in 2002 respectively. CONCLUSION: The resistance of P. falciparum to chloroquine declined progressively after its use had been stopped or reduced in Hainan and Yunnan provinces.

[Factors affecting the in vitro microtest for drug sensitivity of Plasmodium falciparum].

OBJECTIVE: To explore factors influencing the results of in vitro microtest for drug sensitivity of Plasmodium falciparum (Pf). METHODS: Handy media, microplates predisposed with antimalarial drug, cultured Pf parasites (FCC-1/HN isolate) and blood samples from patients were used to evaluate the factors influencing the in vitro determination of drug sensitivity of Pf. RESULTS: Liquid medium and lyophilized medium stored at 4 degrees C for 2 months and 1 year respectively could keep their effect unchanged. The effect of the drug-coated plates was not changed within the following period of storage: plates coated with chloroquine and piperaquine stored at 4 degrees C for 2 years and 6 months respectively; plates coated with pyronaridine and artesunate stored at 4 degrees C for 3 months. The adhesive paper of the sealed plate could be unsealed once only. The plastic plate must be harmless to the growing of parasites. The drug liquid should not be stored over 2 wk at 4 degrees C; otherwise the drug concentration was changed. Parasites tested were at synchronous ring stage, with a density of 1,000-80,000/microliter blood, stored at room temperature for 1 h, and at 4 degrees C for 48 h. Operation needed to follow strictly the standard technical procedure. CONCLUSION: Drug plates, media, adhesive paper, parasites and operation technique can affect the result of in vitro microtest for drug sensitivity of P. falciparum. Standardized materials and operational procedure should be used to guarantee a reliable result of the test.

[The correlation between tooth pain and bioactivators changes in gingival crevicular fluid after applying orthodontic stress].

OBJECTIVE: To investigate the changes of tooth pain in patients with orthodontic tooth movement, to study the changes of bioactivators in GCF in patients with orthodontic tooth movement, and explore the correlation between pain and bioactivators in orthodontic patients. METHODS: 50 patients were included, each having one treatment tooth and one contralateral control tooth. The levels of PGE(2), P substances, IL-6 and GM-CSF in GCF of upper lateral incisor were investigated before activation and at 12th hour, 24th hour, 48th hour and 72th hour after the applying labial orthodontic force, by using highly sensitive radioimmunassay. To record the changes of pain in orthodontic patients at 12th hour, 24th hour,48th hour and 72th hour after applying stress. RESULTS: At experimental sides, total amount of GCF PGE(2), P substances(SP), IL-6 and GM-CSF levels was significantly elevated compared with that before activation. The concentration of PGE(2) and SP in GCF increased significantly and reached peak point at 24th hour in patients, while the levels of IL-6 and GM-CSF in GCF remained at higher baseline through the experiment. There was an rhythm change of periodontal pain during orthodontic tooth movement. CONCLUSION: There was an rhythm changes of pain after stress in patients with orthodontic tooth movement, and the rhythm pain was correlated to the changes of some activator such as P substances and PGE(2) in periodontal ligament.

[Study on treatment of multi-drug resistant falciparum malaria by using a combination of dihydroartemisinin and pyronaridine].

OBJECTIVE: To provide a combined medication scheme for the treatment of multi-drug resistant falciparum malaria. METHODS: Combined administration of dihydroartemisinin and pyronaridine was given to the 32 cases of falciparum malaria cases with multi-drug resistance. The indices for evaluation on day 14, 21, and 28 after treatment included the mean fever subsidence time, mean asexual form clearance time, mean recrudescence time of asexual form and recrudescence rate, proportion of gametocyte carriers, mean density of gametocytes and its mean clearance time, cure rate and rate of side-effects. A double blind clinical test was performed with standard schemes of dihydroartemisinin (20 cases) and pyronaridine (25 cases) as control. RESULTS: The mean fever subsidence time of treated patients by dihydroartemisinin/pyronaridine combination, dihydroartemisinin and pyronaridine was 35.7 +/- 24.7 h, 52.6 +/- 38.9 h and 35.8 +/- 16.5 h respectively, showing a significant difference between the combination group and dihydroartemisinin groups (P < 0.01). The mean asexual form clearance time was 23.8 +/- 10.1 h, 22.9 +/- 6.5 h and 49.4 +/- 20.3 h respectively, showing significantly faster in the combination group than the pyronaridine group (P < 0.01). The recrudescence rate was 0, 4.2% and 0 respectively. The proportion of gametocyte carriers was 20.0%, 16.7% and 60.9% respectively, with a significantly higher rate in the group of pyronaridine than the group of combination (P < 0.01). CONCLUSION: The combination of dihydroartemsinin and pyronaridine is an ideal medication scheme for the treatment of falciparum malaria cases with multi-drug resistance.