Purpose To establish a new method for detecting p16INK4a in cervical tissues with time-resolved fluoroimmunoassay (TRFIA).Methods 126 cases of paraffin imbedding tissues of cervix were selected for immunohistochemistry (IHC) of EnVision two-step and TRFIA.Results There were 20 cases of no intraepithelial lesion or malignancy,24 cases of low-grade squamous intraepithelial lesion (LSIL),53 cases of high-grade squamous intraepithelial lesion (HSIL) and 29 cases of squamous cell carcinoma (SCC).In the groups of no intraepithelial lesion or malignancy,LSIL,HSIL and SCC,p161NK4a positive was seen in 1,19,53 and 28,respectively.TRFIA test results displayed p16INK4a positive in 3,17,50 and 27 cases,respectively.Positive of p16 using by TRFIA in no intraepithelial lesion or malignancy,LSIL,above HSIL was 15.00％,70.83％ and 93.90％,respectively (P ＜ 0.01).Conclusion TRFIA is suitable for detecting of p16INK4a protein and demand low detection equipment,p16INK4a expression detected by TRFIA may helpful for large scale detection in various clinical institution.

Through introducing mutations into ribosomes by obtaining spontaneous drug resistance of microorganisms, ribosome engineering technology is an effective approach to develop mutant strains that overproduce secondary metabolites. In this study, ribosome engineering was used to improve the yield of butenyl-spinosyns produced by Saccharopolyspora pogona by screening streptomycin resistant mutants. The yields of butenyl-spinosyns were then analyzed and compared with the parent strain. Among the mutants, S13 displayed the greatest increase in the yield of butenyl-spinosyns, which was 1.79 fold higher than that in the parent strain. Further analysis of the metabolite profile of S13 by mass spectrometry lead to the discovery of Spinosyn α1, which was absent from the parent strain. DNA sequencing showed that there existed two point mutations in the conserved regions of rpsL gene which encodes ribosomal protein S12 in S13. The mutations occurred a C to A and a C to T transversion mutations occurred at nucleotide pair 314 and 320 respectively, which resulted in the mutations of Proline (105) to Gultamine and Alanine (107) to Valine. It also demonstrated that S13 exhibited genetic stability even after five passages.
Genetic Engineering ; Macrolides ; metabolism ; Point Mutation ; Ribosomal Proteins ; genetics ; Ribosomes ; metabolism ; Saccharopolyspora ; metabolism

Objective: To evaluate the impact of rehabilitation on exercise capacity, cardiopulmonary function and quality of life (QoL) in patients with chronic heart failure (CHF). Methods: A total of 96 CHF patients with NYHA Ⅱ-Ⅲ and left ventricular ejection fraction (LVEF)<40% were enrolled. Based on routine drug therapy, the patients were randomly assigned into 2 groups: Control group, n=50 and Rehabilitation group, n=46, the patients performed treadmill exercise, the training intensity was tailored by (50-80) % of peak oxygen uptake (peak VO2) of baseline cardiopulmonary exercise test (CPET) at (25-40) min/session, 3 sessions/week for 12 weeks. The peak VO2, VE/VCO2 slop, anaerobic threshold (VO2 AT), maximum workload and maximum exercise time were measured by CPET; left atrial diameter (LAD), left ventricular end diastolic diameter (LVEDD), cardiac index (CI) and LVEF were examined by echocardiography; 6 min walking distance (6MWD) and plasma NT-proBNP level were recorded; QoL was assessed by Minnesota living with heart failure questionnaire (MLWHFQ). The above indexes were compared within Rehabilitation group and between 2 groups. Results: In Rehabilitation group, compared to baseline condition, the following indexes were increased by 12 weeks training: peak VO2 (19.8±2.7) ml/min?kg vs (17.4±2.1) ml/min?kg, VO2 AT (11.6±2.5) ml/min?kg vs (9.5±1.8) ml/min?kg, maximum workload (120±20) w vs (102±21) w, maximum exercise time (8.2±1.7) min vs (6.4±1.5) min, CI (2.2±0.5) L/(min?m2) vs (1.9±0.4) L/(min?m2), LVEF (42±5) % vs (35±4) % and 6MWD (406±58) m vs (345±79) m, all P<0.05; while the following parameters were decreased: VE/VCO2 slop (31.7±4.6) vs (34.2±5.8), LAD (38.6±5.5) mm vs (41.5±3.6) mm, LVEDD (58.4±6.3) mm vs (62.9±5.4) mm, NT-proBNP (235±69) ng/ml vs (387±57) ng/ml and MLWHFQ (30.8±12.0) vs (42.3±8.5), all P<0.05. The above indexes were different between Control group and Rehabilitation group, all P<0.05. Conclusion: Rehabilitation may safely and effectively improve cardiopulmonary function and quality of life in CHF patients.

OBJECTIVEIn order to observe the therapeutic efficacy of food control to recurrent oral ulcer (ROU), alimentary control of certain foods was employed to relieve outbreak of ROU.

METHODSThe kits for food intolerant IgG of certain food were used to test the intolerant food of fifty patients with ROU. Observations and assessments for alimentary control were made after three months' treatment on these patients.

RESULTSThe top three of intolerant foods were crab, egg and milk and the remission rate of ROU reached 74% after treatment.

CONCLUSIONThe result of food intolerant IgG testing has certain function to alimentary control therapy for remitting the outbreak of ROU.

Adult ; Diet ; Female ; Food ; Humans ; Male ; Middle Aged ; Oral Ulcer

OBJECTIVETo establish an tachyzoite-brachyzoite interconversion system for Toxoplasma gondii RH strain in vitro.

METHODSCOS-7 cells were inoculated with purified tachyzoites of T.gondii RH strain and cultured in vitro. The morphology of the cultured cells and parasites was observed and the total cellular RNA extracted on days 1 to 6 following the inoculation for detecting the expression of tachyzoite-specific protein (SAG1) and bradyzoite-specific proteins (BAG1 and SAG2C) using RT-PCR.

RESULTSWith the passage of time, the number of parasites in COS-7 cells increased but the proliferation rate was lowered gradually. The intracellular tachyzoites proliferated by means of budding and binary fission, which led to the changes in the alignment of the parasites in the cells from curved pairs, rosette or clustered, and semi-circular patterns to spherical encapsulation-like structures. These changes indicated the gradual transformation of the tachyzoites into bradyzoites. The expressions of the tachyzoite-specific SAG1 gene were detected throughout the 6 days of in vitro culture. The expression of the bradyzoite-specific BAG1 gene had been detected since the second day after the inoculation and SAG2C gene since the fifth day. Alteration of the culture condition resulted in gradual transformation of the bradyzoites into tachyzoites.

CONCLUSIONAn in vitro tachzoites-bradyzoite interconversion system for T.gondii has been successfully established, which provides the basis for further study of the mechanism of interconversion.

Animals ; COS Cells ; Cell Culture Techniques ; Cercopithecus aethiops ; Cysts ; Female ; Genes, Protozoan ; genetics ; Host-Parasite Interactions ; Mice ; Protozoan Proteins ; biosynthesis ; genetics ; Toxoplasma ; growth & development ; physiology

OBJECTIVETo explore the limitation of non-invasive prenatal testing (NIPT) technique through analyzing two false negative cases.

METHODSChromosomal karyotyping analysis was performed on umbilical cord blood sample derived from case 1 at 24 weeks' gestation and peripheral blood sample derived from the neonate of case 2. Placental tissues of case 1 and peripheral blood sample of case 2 were also analyzed by high-throughput sequencing for copy number variations (CNVs).

RESULTSFor case 1, analysis of fetal umbilical cord blood sample showed a translocation type of trisomy 21, i.e., 46,XY,der(21;21)(q10;q10),+21. There were no obvious abnormalities detected at or near the center of the fetal surface and matrix surface of the placenta. High-thoroughput sequencing showed Chr13:(33 840 001 - 115 100 000)×3[60%]/46,XY[40%] at the edge of the placenta, Chr13:(34 080 001-115100000)×3[54%]/46,XY[46%] at the edge of placenta matrix surface, and trisomy 21 in the umbilical cord tissue. For case 2, analysis of the neonatal peripheral blood sample showed a karyotype of 46,XY,del(18)(q22), which revealed a microdeletion in chromosome 18. High-throughput sequencing of the maternal peripheral blood sample stored during pregnancy confirmed it to be chr18: (62 910 000 - 78 020 000)×1 with 15.1 Mb deletion in the fetus. The neonate was therefore diagnosed with partial monosomy of chromosome 18.

CONCLUSIONFalse negative results of NIPT are related with the fraction of circulating cell-free fetal DNA in the maternal serum. NIPT has limitations in detecting fetal chromosomal microdeletion and confined placenta mosaicisms. Routine ultrasound scan is necessary for pregnant women with low-risk indicated by NIPT.

Adult ; Aneuploidy ; Chromosomes, Human, Pair 18 ; Diagnostic Errors ; Female ; High-Throughput Nucleotide Sequencing ; Humans ; Karyotyping ; Monosomy ; Pregnancy ; Prenatal Diagnosis ; methods

Menkes disease is a rare X-linked recessive disorder characterized by multi-systemic disorder of copper deficiency caused by ATP7A gene mutation. In this study, the clinical and laboratory features of three patients with Menkes disease were analyzed. Prenatal diagnosis had been performed for a fetus of a family. Three patients were admitted at the age of 8-9 months due to severe epilepsies and marked delayed psychomotor development. Significantly light complexion, pudgy cheeks and sparse fuzzy wooly hair were observed. On their cranial MR imaging, cortical atrophy, leukoencephalopathy, basal ganglia damage and tormesity of the intracranial vessels were found. Their plasma ceruloplasmin decreased to 70.2, 73.5 and 81 mg/L, significantly lower than normal range (210-530 mg/L). c.3914A>G (p. D1305G) was detected on ATP7A gene of case 1 and 2. A novel mutation, c.3265G>T (p.G1089X) was found in case 3. Both of them were firstly found in Chinese patients of Menkes disease. The mother of case 1 was tested at 20 weeks of pregnancy. Karyotype and ATP7A gene studies of the amniocytes were performed for the prenatal diagnosis of her fetus. Normal male karyotypes without c.3914A>G mutation on ATP7A gene was showed. Postnatal genetic analysis and normal development confirmed the prenatal diagnosis.
Adenosine Triphosphatases ; genetics ; Cation Transport Proteins ; genetics ; Copper-transporting ATPases ; Humans ; Infant ; Male ; Menkes Kinky Hair Syndrome ; diagnosis ; genetics ; Mutation ; Prenatal Diagnosis

This study was aimed to explore the potential therapy of Gambogic acid (GA) combined with magnetic nanoparticle of Fe3O4 (Fe3O4-MNP) on leukemia. The proliferation of U937 cells and the cytotoxicity were evaluated by MTT assay. Cell apoptosis was observed and analyzed by microscopy and flow cytometry respectively. The expressions of gene and protein were detected by quantitative real-time polymerase chain reaction and Western blot respectively. The results showed that GA enhanced the cytotoxicity for U937 cells in dose- and time-dependent manners. The Fe3O4-MNP itself had not cytotoxicity, but could enhance the inhibitory effect of GA on proliferation of U937 cells. The apoptotic rate of U937 cells induced by combination of GA with Fe3O4-MNP was higher than that by GA alone. The typical apoptotic features of cells treated with GA and Fe3O4-MNP were observed. The expression levels of caspase-3 and bax after co-treatment of GA and Fe3O4-MNP were higher than that exposed to GA or Fe3O4-MNP alone, but the expressions of bcl-2, NF-kappaB and survivin were down-regulated. It is concluded that Fe3O4-MNP can promote GA-induced apoptosis in U937 cells, and the combination of GA with Fe3O4-MNP may be a safer and less toxic new therapy for leukemia.
Apoptosis ; drug effects ; Humans ; Iron Compounds ; administration & dosage ; pharmacology ; Magnetics ; Nanoparticles ; U937 Cells ; Xanthones ; pharmacology

As extremely important inflammatory cells in the tumor microenvironment, tumor-associated macrophages (TAMs) can secrete a variety of chemokines and cytokines, which play an important role in the occurrence of tumor growth and metastasis. Recent years, increasing studies have shown that macrophages are associated with tumor chemotherapy sensitivity. The chemical substances produced by macrophages affect the efficacy of chemotherapeutic agents. In addition, some chemotherapeutic agents have an effect on the recruitment and bioactivity of macrophages in the tumor issue, which influences the anti-tumor efficacy of chemotherapy drugs. In this review, we summarize the roles of macrophages in the chemotherapy resistance, including the regulatory mechanism and the strategy of targeting macrophages.

Objective: To determine the effect of the combination of lapatinib with chlorogenic acid on metastasis of breast cancer in mouse model . Methods: The classical macrophage M2 polarization model induced by interlukin 13 in vitro was adopted in the study .Flow cytometric analysis was performed to detect the expression of M2 marker CD206 . The transcription of M2-associated genes was measured by RT-PCR.HE staining was used to analyze the metastatic nodes of breast cancer in lungs of MMTV-PyVT mice.Immunostaining analysis was used to detect the expression of related proteins in breast cancer .Results:The combination of lapatinib and chlorogenic acid inhibited the expression of CD206 induced by IL-13 [(42.17% ±2.59%) vs ( 61 .15%±7 .58%) , P<0 .05 ] . The combination more markedly suppressed expression of M2-associated gene Ym1 than lapatinib alone [(0.9 ±0.1) vs (1.8 ± 0 .0 ) , P <0 .05 ] .The combination of lapatinib and chlorogenic acid significantly reduced metastatic nodes in lung [ P <0 .05 ] , and also significantly decreased the percentage of CD206 +cells in breast cancer compared to controls [(6.08%±2.60%) vs ( 29 .04%±5 .86%) , P<0 .05 ] .Conclusion: The combination of lapatinib and chlorogenic acid can effectively inhibit macrophage M 2 polarization and metastasis of breast cancer .