从构建的噬菌体抗体库中筛选抗甲肝人单抗

用固相化甲肝抗原,对所构建的噬菌体抗体库进行了3轮淘筛。第3轮淘筛后洗脱下来的噬菌体,较第l轮增加了近100倍。含有抗体重链基因和轻链基因的重组克隆．也由淘筛前的25％增至100％。说明甲肝抗原对抗体库的淘筛,富集了表面呈现甲肝人单抗的噬菌体。经夹心ELISA法筛选到抗甲肝病毒噬菌体抗体,并以竞争抑制实验进一步证实了这些抗体的特异性。     

Overcoming resistance to rapalogs in gliomas by combinatory therapies

Glioblastoma is the most common and aggressive brain tumor type, with a mean patient survival of approximately 1 year. Many previous analyses of the glioma kinome have identified key deregulated pathways that converge and activate mammalian target of rapamycin (mTOR). Following the identification and characterization of mTOR-promoting activity in gliomagenesis, data from preclinical studies suggested the targeting of mTOR by rapamycin or its analogs (rapalogs) as a promising therapeutic approach. However, clinical trials with rapalogs have shown very limited efficacy on glioma due to the development of resistance mechanisms. Analysis of rapalog-insensitive glioma cells has revealed increased activity of growth and survival pathways compensating for mTOR inhibition by rapalogs that are suitable for therapeutic intervention. In addition, recently developed mTOR inhibitors show high anti-glioma activity. In this review, we recapitulate the regulation of mTOR signaling and its involvement in gliomagenesis, discuss mechanisms resulting in resistance to rapalogs, and speculate on strategies to overcome resistance. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).     

Probing the ligand preferences of the three types of bacterial pantothenate kinase

Pantothenate kinase (PanK) catalyzes the transformation of pantothenate to 4′-phosphopantothenate, the first committed step in coenzyme A biosynthesis. While numerous pantothenate antimetabolites and PanK inhibitors have been reported for bacterial type I and type II PanKs, only a few weak inhibitors are known for bacterial type III PanK enzymes. Here, a series of pantothenate analogues were synthesized using convenient synthetic methodology. The compounds were exploited as small organic probes to compare the ligand preferences of the three different types of bacterial PanK. Overall, several new inhibitors and substrates were identified for each type of PanK.     

Cell Size and Growth Rate Are Modulated by TORC2-Dependent Signals

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Treatment of donor cells with trichostatin A improves in vitro development and reprogramming of buffalo (Bubalus bubalis) nucleus transfer embryos

It has been reported that buffalo (Bubalus bubalis) embryos reconstructed by somatic cell nucleus transfer (SCNT) can develop to the full term of gestation and result in newborn calves. However, the developmental competence of reconstructed embryos is still low. Recently, it has been reported that treating donor cells or embryos with trichostatin A (TSA) can increase the cloning efficiency in some species. Thus, the present study was undertaken to improve the development of buffalo SCNT embryos by treatment of donor cells (buffalo fetal fibroblasts) with TSA and explore the relation between histone acetylation status of donor cells and developmental competence of SCNT embryos. Treatment of donor cells with either 0.15 or 0.3 μM TSA for 48 hours resulted in a significant increase in the cleavage rate and blastocyst yield of SCNT embryos (P < 0.05). Meanwhile, the expression level of HDAC1 in donor cells was also decreased (0.4–0.6 fold, P < 0.05) by TSA treatment, although the expression level of HAT1 was not affected. Further measurement of the epigenetic maker AcH4K8 in buffalo IVF and SCNT embryos at the eight-cell stage revealed that the spatial distribution of acH4K8 staining in SCNT embryos was different from the IVF embryos. Treatment of donor cells with TSA resulted in an increase in the AcH4K8 level of SCNT embryos and similar to fertilized counterparts. These results suggest that treatment of donor cells with TSA can facilitate their nucleus reprogramming by affecting the acetylated status of H4K8 and improving the in vitro development of buffalo SCNT embryos. The AcH4K8 status at the eight-cell stage can be used as an epigenetic marker for predicting the SCNT efficiency in buffalos.     

Temperature-jump NMR study of protein folding: Ribonuclease A at low pH

Summary The kinetic process of folding of bovine pancreatic ribonuclease A in a²H₂O environment at pH 1.2 was examined by a recently developed temperature-jump NMR method (Akasaka et al., (1990) Rev. Sci. Instrum.61, 66–68). Upon temperature-jump down from 45°C to 29°C, which was attained within 6 s, the proton NMR spectral changes were followed consecutively in time intervals of seconds. There was a rapid spectral change, which was finished within the jump period, followed by a much slower process which lasted for a minute or longer. Rates of the slower process were measured at different positions of the polypeptide chain as intensity changes of individual His and Tyr proton signals of the folded conformer and as intensity changes of aliphatic and His protons of the unfolded conformer. Most of these rates coincided with each other within experimental error with an average value of 2.8×10^–2s^–1. The result gave clear experimental evidence that the slow folding of RNase A at low pH is a cooperative process involving most regions of the molecule, not only thermodynamically, but kinetically as well.     

Next generation sequencing demonstrates association between tumor suppressor gene aberrations and poor outcome in patients with cancer

Next generation sequencing is transforming patient care by allowing physicians to customize and match treatment to their patients’ tumor alterations. Our goal was to study the association between key molecular alterations and outcome parameters. We evaluated the characteristics and outcomes (overall survival (OS), time to metastasis/recurrence, and best progression-free survival (PFS)) of 392 patients for whom next generation sequencing (182 or 236 genes) had been performed. The Kaplan-Meier method and Cox regression models were used for our analysis, and results were subjected to internal validation using a resampling method (bootstrap analysis). In a multivariable analysis (Cox regression model), the parameters that were statistically associated with a poorer overall survival were the presence of metastases at diagnosis (P = 0.014), gastrointestinal histology (P < 0.0001), PTEN (P < 0.0001), and CDKN2A alterations (P = 0.0001). The variables associated with a shorter time to metastases/recurrence were gastrointestinal histology (P = 0.004), APC (P = 0.008), PTEN (P = 0.026) and TP53 (P = 0.044) alterations. TP53 (P = 0.003) and PTEN (P = 0.034) alterations were independent predictors of a shorter best PFS. A personalized treatment approach (matching the molecular aberration with a cognate targeted drug) also correlated with a longer best PFS (P = 0.046). Our study demonstrated that, across diverse cancers, anomalies in specific tumor suppressor genes (PTEN, CDKN2A, APC, and/or TP53) were independently associated with a worse outcome, as reflected by time to metastases/recurrence, best PFS on treatment, and/or overall survival. These observations suggest that molecular diagnostic tests may provide important prognostic information in patients with cancer.     

Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.     

An engram found? Evaluating the evidence from fruit flies

Is it possible to localize a memory trace to a subset of cells in the brain? If so, it should be possible to show: first, that neuronal plasticity occurs in these cells. Second, that neuronal plasticity in these cells is sufficient for memory. Third, that neuronal plasticity in these cells is necessary for memory. Fourth, that memory is abolished if these cells cannot provide output during testing. And fifth, that memory is abolished if these cells cannot receive input during training. With regard to olfactory learning in flies, we argue that the notion of the olfactory memory trace being localized to the Kenyon cells of the mushroom bodies is a reasonable working hypothesis.