Proposed flow cytometric reference method for the determination of erythroid F-cell counts

BACKGROUND: Quantitation of adult erythrocytes (RBC) containing fetal hemoglobin (F cells) is of potential clinical utility in evaluating erythropoietic disorders, such as myelodysplasia and hemoglobinopathies, and in monitoring F-cell augmenting therapy. F-cell counting methodologies include fluorescence microscopy and flow cytometry. Previous flow cytometric methods have employed an isotype antibody control to distinguish F cells from non-F cells. We investigated the feasibility of using the orange autofluorescence signal (FL2) in glutaraldehyde-fixed RBC to substitute for fluorescein isothiocyanate (FITC)-labeled isotype control antibody use in F-cell quantitation. METHODS: Our previously published method for fetal red cell detection in fetomaternal hemorrhage was used, employing a FITC-labeled anti-hemoglobin F (HbF) monoclonal antibody reagent. Blood samples with varying F-cell counts were quantitated for F cells using both immunofluorescence microscopy and flow cytometry comparing FITC-labeled isotype to FL1 thresholding defined by FL2 autofluorescence. RESULTS: F cell percentages obtained by using an FL2 defined threshold for FL1 gating correlated well with expected values in diluted blood samples (r(2) = 0.994, slope = 1. 019, intercept = 0.24), values obtained using an isotype control (r(2) = 0.996, slope = 1.012, intercept = -0.17), and microscopic immunofluorescence counts (r(2) = 0.989, slope = 0.999, intercept = -0.72). F-cell quantitation by the isotype control and FL2 autofluorescence methods was also comparable in 40 blood samples (r(2) = 0.994, slope = 1.014, intercept = 0.03). Intra-assay, interobserver, and interinstrument precision with this autofluorescence gating method exhibited low imprecision (coefficient of variation <14%). CONCLUSION: This novel method is a more objective and less laborious alternative for F-cell quantitation by flow cytometry compared to using an isotype control or microscopy, thereby providing a more robust methodology for clinical studies and consideration as a laboratory reference method for F-cell counting.     

The noise of membrane capacitance measurements in the whole-cell recording configuration

High-resolution measurement of membrane capacitance in the whole-cell-recording configuration can be used to detect small changes in membrane surface area that accompany exocytosis and endocytosis. We have investigated the noise of membrane capacitance measurements to determine the fundamental limits of resolution in actual cells in the whole-cell mode. Two previously overlooked sources of noise are particularly evident at low frequencies. The first noise source is accompanied by a correlation between capacitance estimates, whereas the second noise source is due to "1/f-like" current noise. An analytic expression that summarizes the noise from thermal and 1/f sources is derived, which agrees with experimental measurements from actual cells over a large frequency range. Our results demonstrate that the optimal frequencies for capacitance measurements are higher than previously believed. Finally, we demonstrate that the capacitance noise at high frequencies can be reduced by compensating for the voltage drop of the sine wave across the series resistance.     

RNAi介导的OsBTF3基因沉默及其对水稻籽粒相关性状的影响

为了创制OsBTF3基因沉默的水稻植株、验证该基因在水稻籽粒相关性状中的功能、评价其在水稻遗传改良中潜在的应用价值,设计和合成OsBTF3基因序列的引物、扩增部分基因片段,构建RNAi基因沉默载体、通过农杆菌介导转化愈伤组织、植株再生、潮霉素抗性筛选和PCR验证、定量分析OsBTF3基因表达量,测定转基因水稻籽粒相关性状。结果表明,成功地获得了20个T1代OsBTF3-RNAi转基因株系,OsBTF3基因表达量得到显著的抑制和干扰,抑制效果平均达到85%;与野生型对照株相比,5个所测定RNAi转基因株系的穗长、穗粒数、千粒重和穗粒重等籽粒相关性状明显地减小或降低。因此,RNAi介导的基因沉默导致了OsBTF3基因表达水平抑制以及在籽粒性状中的功能缺失;OsBTF3可能是一个调控水稻籽粒相关性状重要的功能基因。     

Generation of Hepatitis B virus PreS2-S antigen in Hansenula polymorpha

Despite the long availability of a traditional prophylactic vaccine containing the HBV surface antigen(HBsA g) and aluminum adjuvant, nearly 10% of the population remains unable to generate an effective immune response. Previous studies have indicated that hepatitis B virus(HBV) PreS 2-S is abundant in T/B cell epitopes, which induces a stronger immune response than HBsA g, particularly in terms of cytotoxic T lymphocyte(CTL) reaction. In the current study, the HBV PreS 2-S gene encoding an extra26 amino acids(PreS 2 C-terminus) located at the N-terminus of HBsA g was cloned into the pV CH1300 expression vector. Pre S2-S expressed in the methylotrophic yeast, Hansenula polymorpha, was produced at a yield of up to 250 mg/L. Subsequent purification steps involved hydrophobic adsorption to colloidal silica, ion-exchange chromatography and density ultracentrifugation. The final product was obtained with a total yield of ~15% and purity of ~99%. In keeping with previous studies, ~22 nm viruslike particles were detected using electron microscopy. The generated PreS 2-S antigen will be further studied for efficacy and safty in animals.     

Acyclic nucleoside/nucleotide analogues with an imidazole ring skeleton

Syntheses of a few acyclic nucleoside and acyclic nucleoside phosphonate analogues containing an imidazole ring have been reported. These analogues include methyl 1-(2-hydroxyethoxymethyl)imidazole-4, 5-dicarbo-xylate (1), 4,5-dicarbamoyl-1-(2-hydroxyethoxymethyl)imidazole (2), 4,5-dicyano-1-(2-hydroxyethoxymethyl)imidazole (4), Methyl 1-(2-bromoethoxymethyl)imidazole-4,5-dicarboxylate (7), 4,5-dicyano-(2-bromoethoxymethyl)imidazole (8), and Methyl 1-(2-phosphonomethoxyethyl)imidazole (10). Also reported are a few potential prodrugs of the above compounds, including the acetyl derivatives 5 and 6 (of 1 and 4, respectively), and the diethyl phosphonate ester 9 (of 10). In addition, the corresponding benzyl-protected precursors 11 and 12 (of 1 and 4, respectively), along with their common hydrolysis product, 1-(2-benzyloxy-ethoxymethyl)-4,5-imidazoledicarboxylic acid (3), are reported. Another potential prodrug included in the list is 1-(2-acetoxyethyl)-4,5-dicyanoimidazole (15). The compounds were screened for in vitro antiviral activity against a wide variety of herpes and respiratory viruses. The most active compound was the phosphonate analogue 9 which exhibited an anti-measles virus activity with an EC50 of <2.5 microg/mL and an SI value of > 176.     

昆虫神经生物学研究技术:触角电位图记录

触角电位图记录是昆虫神经生物学中用于昆虫嗅觉、信息素生物测定研究的重要技术之一。该技术具有敏感性、选择性高及操作简单易行等优点。该文以荷兰Syntech公司的产品为例详细介绍了此技术原理、装置、操作及应用     

Structures and magnetic properties of mono-doped fullerenes C59Xn and C59X(6mn)m (X=Bm,N+, P+, As+, Si): isoelectronic analogues of C60 and C60(6m)

Structures of mono-doped fullerenes, C59Xn and C59X(6mn)m (X=Bm, N+, P+, As+, Si), the isoelectronic analogues to C60 and C606m with 60 and 66 pi-electrons, have been investigated at the B3LYP/6-31G* level of density functional theory. On the basis of the computed nucleus independent chemical shifts (NICS) at the cage center and also at the center of individual rings as magnetic criteria, heterofullerenes with 60 pi-electrons are as aromatic as the parent C60, while those with 66 pi-electrons are much less aromatic than C606m. The very distinct endohedral chemical shifts of the 66 pi-electron systems may be useful to identify the heterofullerenes through their endohedral 3He NMR chemical shifts.     

CCL5/CCR5 axis promotes the motility of human oral cancer cells

CCL5 (previously called RANTES) is in the CC‐chemokine family and plays a crucial role in the migration and metastasis of human cancer cells. On the other hand, the effect of CCL5 is mediated via CCR receptor. RT‐PCR and flow cytometry studies demonstrated CCR5 but not CCR1 and CCR3 mRNA in oral cancer cell lines, especially higher in those with high invasiveness (SCC4) as compared with lower levels in HSC3 cells and SCC9 cells. Stimulation of oral cancer cells with CCL5 directly increased the migration and metalloproteinase‐9 (MMP‐9) production. MMP‐9 small interfering RNA inhibited the CCL5‐induced MMP‐9 expression and thereby significantly inhibited the CCL5‐induced cell migration. Activations of phospholipase C (PLC), protein kinase Cδ (PKCδ), and NF‐κB pathways after CCL5 treatment was demonstrated, and CCL5‐induced expression of MMP‐9 and migration activity was inhibited by the specific inhibitor of PLC, PKCδ, and NF‐κB cascades. In addition, migration‐prone sublines demonstrate that cells with increasing migration ability had more expression of MMP‐9, CCL5, and CCR5. Taken together, these results indicate that CCL5/CCR5 axis enhanced migration of oral cancer cells through the increase of MMP‐9 production. J. Cell. Physiol. 220: 418–426, 2009. © 2009 Wiley‐Liss, Inc.