RESUMO
OBJECTIVE: To investigate the appropriate nanomanipulation of slides of human tongue squamous cell carcinoma tissue and cultured cells by atomic force microscopy (AFM), and develop methods to improve the resolution and contrast of AFM images. METHODS: Human tongue squamous carcinoma cells of the line Tca8113 were cultured. Cell masses were isolated and collected, and sectioned. Then the white lead and silvery white sections were transferred on mica slides, dried. Eighty slides were used and divided into 2 groups to be immersed into hydrogen dioxide of the concentrations 20% and 30% for 0, 5, 10, 15, 20, 25, 30, and 40 min respectively. Samples of human tongue squamous carcinoma were collected from 15 cases. Every sample was divided into 2 halves. Half of every sample underwent routine paraffin preparation of slide and adhered onto glass, dissolving of paraffin with dimethylbenzene for 0, 10, 15, 30, 45, or 60 min; and half of the sample underwent routine preparation of slides for electron microscopy, the white lead and silvery white sections were transferred on mica slides, dried. The slides were observed by contact mode AFM in air. RESULTS: In the ultrathin sections the metastructure of the cell such as the cell membrane, nuclear membrane and nucleolus could be distinct from each other clearly. The profile of the cell and the nucleus inside could be revealed by AFM after the paraffin sections were treated in dimethylbenzene for 15-30 min. AFM manipulation could be achieved within nanometer range by precise modulation of scanning force, area, angle and so on. The appropriate needle point should contain elastic coefficient > 5.0 N/m. The force exerted on the needle point should be within the range 50-300 nN. After location the scanning direction should be within the range of 20 nm-1 microm. CONCLUSION: A series of modified techniques of preparing and treating AFM samples, such as adhering the sections onto mica slide, dissolving of epoxy resin by hydrogen dioxide and removal of paraffin by dimethylbenzene, were developed, thus realizing high resolving power AFM imaging, especially for nuclear membrane, and nucleolus, and providing a base of AFM location, dissection, isolation and obtainment at nanometer scale.
Assuntos
Carcinoma de Células Escamosas/patologia , Microscopia de Força Atômica/métodos , Nanotecnologia/métodos , Neoplasias da Língua/patologia , Humanos , Células Tumorais CultivadasRESUMO
The [-2]proPSA (p2PSA) and its derivatives, the p2PSA-to-free PSA ratio (%p2PSA), and the Prostate Health Index (PHI) have greatly improved discrimination between men with and without prostate cancer (PCa) in prostate biopsies. However, little is known about their performance in cases where a digital rectal examination (DRE) and transrectal ultrasonography (TRUS) are negative. A prospective cohort of 261 consecutive patients in China with negative DRE and TRUS were recruited and underwent prostate biopsies. A serum sample had collected before the biopsy was used to measure various PSA derivatives, including total prostate-specific antigen (tPSA), free PSA, and p2PSA. For each patient, the free-to-total PSA ratio (%fPSA), PSA density (PSAD), p2PSA-to-free PSA ratio (%p2PSA), and PHI were calculated. Discriminative performance was assessed using the area under the receiver operating characteristic curve (AUC) and the biopsy rate at 91% sensitivity. The AUC scores within the entire cohort with respect to age, tPSA, %fPSA, PSAD, p2PSA, %p2PSA, and PHI were 0.598, 0.751, 0.646, 0.789, 0.814, 0.808, and 0.853, respectively. PHI was the best predictor of prostate biopsy results, especially in patients with a tPSA of 10.1-20 ng ml-1 . Compared with other markers, at a sensitivity of 91%, PHI was the most useful for determining which men did not need to undergo biopsy, thereby avoiding unnecessary procedures. The use of PHI could improve the accuracy of PCa detection by predicting prostate biopsy outcomes among men with a negative DRE and TRUS in China.
Assuntos
Próstata/patologia , Neoplasias da Próstata/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Exame Retal Digital , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Próstata/diagnóstico por imagem , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/patologia , Ultrassonografia/métodosRESUMO
The radial compression properties of single DNA molecules have been studied using vibrating scanning polarization force microscopy. By imaging DNA molecules at different vibration amplitude set-point values, we obtain the correlations between radially applied force and DNA compression, from which the radial compressive elasticity can be deduced. The estimated elastic modulus is approximately 20-70 MPa under small external forces (<0.4 nN) and increases to approximately 100-200 MPa for large loads.
Assuntos
DNA/química , DNA/ultraestrutura , Micromanipulação/métodos , Microscopia de Força Atômica/métodos , Microscopia de Polarização/métodos , Modelos Químicos , Modelos Moleculares , Força Compressiva , Simulação por Computador , DNA/análise , Elasticidade , Interpretação de Imagem Assistida por Computador/métodos , Conformação de Ácido Nucleico , Estresse Mecânico , VibraçãoRESUMO
The apoptotic pathway is important in the control of vital processes of hepatocellular carcinoma (HCC). In the current study, we aimed to determine whether apoptotic gene-related polymorphisms modified HCC prognosis. We genotyped 16 single nucleotide polymorphisms (SNPs) in 10 core genes (TP53, TP53INP1, TP53BP1, CDKN2A, CDKN1A, CDKN1B, MDM2, BAX, CCDN1 and BCL2) in the apoptotic pathway by using DNA from blood samples of 362 HCC patients receiving surgical resection of HCC tumor. The associations between genotypes/haplotypes of the 10 genes and overall survival (OS) of HCC patients were assessed using the Cox proportional hazards model. We found one CDKN1B haplotype CCT/ACT (constructed by rs36228499 C>A, rs34330 C>T and rs2066827 T>G) significantly associated with decreased OS of HCC patients, compared to the common haplotype ACT/CTT both in univariate analysis (P=0.013, HR=1.198, 95% CI: 1.039-1.381) and multivariate analysis (P=0.006, HR=1.224, 95% CI: 1.059-1.413). We also find two SNPs (rs560191 G>C and rs2602141 T>G) in TP53BP1 shown to be marginally significantly associated with decreased OS of HCC patients. However, none of the other SNPs or haplotypes were significantly associated with HCC OS. Our results illustrated the potential use of CDKN1B haplotype as a prognostic marker for HCC patients with surgical resection of tumor.
RESUMO
The genes of short armed hammerhead ribozyme targeting against two sites on positive strand (194-196) and negative strand (89-91) of ASSVd were designed, synthesized and cloned according to the action manner of hammerhead ribozyme. The full lengths of the genes are 42 bp (RzASSVd(+)) and 40 bp (RzASSVd(-)). After transcription in vitro, the ASSVd positive and negative RNA labeled with 32P were mixed with the ribozyme transcript and incubated 3-4 h at 50 degrees C or 37 degrees C. The results were assayed on 8% PAGE (containing 8 mol/L urea) and autoradiogrammed. As predicted, the transcript of the active RzASSVd(-) could cleave the ASSVd negative strand RNA with a high activity but had no cleavage effect on the ASSVd positive strand. The transcript of the RzASSVd(+) gene could cleave the ASSVd positive strand but its cleavage activity was very low. As the same, it cannot cleave the negative strand either. On the base of the result, we construct dimmer ribozyme gene pGEMRzASSVd(+/-) containing both RzASSVd(+) and RzASSVd(-).
Assuntos
Malus/virologia , RNA Catalítico/genética , RNA Viral/metabolismo , Viroides/metabolismo , Clonagem Molecular , Doenças das Plantas/virologia , RNA Catalítico/metabolismo , RNA Catalítico/uso terapêutico , Especificidade por Substrato , Transcrição GênicaRESUMO
A self-cleaving hammerhead ribozyme gene containing a 14nt target sequence of ASSVd at the 3' end of hammerhead ribozyme was synthesized, amplified and cloned at the Xho I-Hind III site of pGEM7Zf(+). The ends produced by Xho I or Sal I can link together, thus the recognition sites of both enzymes vanish and can't be cut by either one. We used this property to get the recombinant plasmid bearing 2, 4, 6, 8, 10 and 12 copies of self-cleavable ribozyme respectively after successively sub-cloning five times. Linearized recombinat plasmid model catalyzed by T7 RNA polymerase was transcribed in vitro. The multimeric ribozyme molecules efficiently self-cleaved via cis-acting to release many ribozyme molecules It indicates that the concentration of ribozyme transcripts has been enhanced during transcription. Trans-cleavage reaction was carried out by incubating monomeric and multimeric ribozymes with same mol concentration and 32P labeled target ASSVd. Both ribozymes and target transcripts were mixed in 1:1 ratio. Autoradiograms showed the transcripts of multimeric ribozyme were substantially more effective against the ASSVd target RNA than the monomeric ribozymes. We confer that the multimeric self-clevable ribozyme is likely to provide more valuable application in vivo.
Assuntos
Malus/virologia , RNA Catalítico/genética , RNA Viral/metabolismo , Viroides/metabolismo , RNA Catalítico/química , RNA Catalítico/metabolismoRESUMO
Ryanodine receptors (RyRs) of skeletal muscle, as calcium release channels, have been found to form semicrystalline arrays in the membrane of sarcoplasmic reticulum. Recently, both experimental observations and theoretical simulations suggested cooperative coupling within interlocking RyRs. To better understand the interactions between RyRs and their modulation, the aggregation and dissociation of isolated RyRs in aqueous medium containing various Na(+) and K(+) concentrations were investigated using photon correlation spectroscopy (PCS) and atomic force microscopy (AFM). RyRs aggregated readily at low salt concentrations. However, a different behavior was observed in the presence of Na(+) or K(+). Detectable aggregates were formed in 5 microg/mL RyR sample when the concentration of Na(+) and K(+) was reduced from 1 M to below 0.28 and 0.23 M, respectively. The dissociation of RyR aggregates was also examined when raising the salt concentration. While aggregates formed in 0.15 M NaCl medium could reverse almost completely, those formed in 0.15 M KCl medium only dissolved partly. When keeping the total salt concentration at 0.15 M, the aggregation and dissociation of RyRs were seen to evidently depend on the relative concentration of Na(+) and K(+). The interaction between RyRs was strengthened with increasing Na(+)/K(+) ratios in the mixed medium. Accompanying this, a decrease of [(3)H]ryanodine binding occurred. The results obtained with PCS and AFM provide further evidence for the interaction between RyRs and suggest the importance of Na(+), K(+), and their relative composition in modulating the interaction and cooperation between RyRs in vivo.