Molecular cloning and sequence analysis of methionine adenosyltransferase from the economic seaweed Undaria pinnatifida

The methionine adenosyltransferase gene (MAT) had been isolated from an economic seaweed Undaria pinnatifida by PCR using degenerate primers. The cDNA was 1,491 bp in length with an open reading frame of 1,194 nucleotides, encoding a deduced protein of 397 amino acids. The protein had a predicted molecular weight of 43.2 kDa, and the isoelectric point was 5.244. The sequence contains a 92 bp 5′-untranslated region (UTR) and a 205 bp 3′-UTR. The methionine adenosyltransferase (MAT) sequence of U. pinnatifida (UpMAT) shared 68–92 % identities with the previous published MAT sequences of other species. Phylogenetic analysis indicated that the phylogenetic relationship of UpMAT with some other seaweeds was closer than with those of higher plants. Under different stress conditions, the relative mRNA expression levels of the MAT of U. pinnatifida (UpMAT) were measured by real-time quantitative PCR, and the results demonstrated that the UpMAT might help to protect the alga against various abiotic stresses.     

金荞麦果实中有效成分的分析

采用HPLC法对金荞麦果实中的有效成分进行了分析,结果表明:金荞麦果实中含有芦丁、槲皮素和表儿茶素等有效成分,其中主要以芦丁为主,槲皮素、表儿茶素等含量甚微.金荞麦果实中的有效成分主要集中在种子部分,果皮中则不合或含量甚微.     

Polymorphisms in MHC class Ia genes and resistance to IHNV in rainbow trout (Oncorhynchus mykiss)

Infectious haematopoietic necrosis virus (IHNV) is detrimental to the farming of rainbow trout (Oncorhynchus mykiss) and other salmonids in the Northern hemisphere. The major histocompatibility complex (MHC) plays a key role in immune response in invertebrates, as evidenced by the close correlation of MHC polymorphisms with disease resistance/susceptibility. To analyse the correlation between rainbow trout resistance and susceptibility to IHNV and genetic variation in exon 2 of MHC class Ia gene, UBA, we employed two approaches, namely, polymerase chain reaction-single strand conformation polymorphism analysis and cloning/sequencing. From 102 resistant and 82 susceptible individuals, a total of 12 alleles in UBA exon 2 (GenBank: JX136662–JX136673) were identified, including 11 novel alleles. The maximum number of these alleles in a single individual was four, suggesting that UBA exon 2 most likely resides on at least two loci in the genome. Most of the variations in UBA exon 2 were located in the peptide-binding region and were determined to have been subject to positive selection during evolution. Correlation analysis revealed that Onmy-UBA*0111 and Onmy-UBA*0107 are highly associated with IHNV susceptibility (P = 0.001), whereas Onmy-UBA*0101, Onmy-UBA*0102, and Onmy-UBA*0103 are highly related to IHNV resistance (P = 0.000). In addition, the three resistant alleles were predominant in the IHNV disease-resistant population; thus, these molecular markers can be used for anti-IHNV breeding of rainbow trout.     

Indirect and suboptimal control of gene expression is widespread in bacteria

Gene regulation in bacteria is usually described as an adaptive response to an environmental change so that genes are expressed when they are required. We instead propose that most genes are under indirect control: their expression responds to signal(s) that are not directly related to the genes’ function. Indirect control should perform poorly in artificial conditions, and we show that gene regulation is often maladaptive in the laboratory. In Shewanella oneidensis MR‐1, 24% of genes are detrimental to fitness in some conditions, and detrimental genes tend to be highly expressed instead of being repressed when not needed. In diverse bacteria, there is little correlation between when genes are important for optimal growth or fitness and when those genes are upregulated. Two common types of indirect control are constitutive expression and regulation by growth rate; these occur for genes with diverse functions and often seem to be suboptimal. Because genes that have closely related functions can have dissimilar expression patterns, regulation may be suboptimal in the wild as well as in the laboratory.     

Determination of Minimum Training Sample Size for Microarray-Based Cancer Outcome Prediction–An Empirical Assessment

The promise of microarray technology in providing prediction classifiers for cancer outcome estimation has been confirmed by a number of demonstrable successes. However, the reliability of prediction results relies heavily on the accuracy of statistical parameters involved in classifiers. It cannot be reliably estimated with only a small number of training samples. Therefore, it is of vital importance to determine the minimum number of training samples and to ensure the clinical value of microarrays in cancer outcome prediction. We evaluated the impact of training sample size on model performance extensively based on 3 large-scale cancer microarray datasets provided by the second phase of MicroArray Quality Control project (MAQC-II). An SSNR-based (scale of signal-to-noise ratio) protocol was proposed in this study for minimum training sample size determination. External validation results based on another 3 cancer datasets confirmed that the SSNR-based approach could not only determine the minimum number of training samples efficiently, but also provide a valuable strategy for estimating the underlying performance of classifiers in advance. Once translated into clinical routine applications, the SSNR-based protocol would provide great convenience in microarray-based cancer outcome prediction in improving classifier reliability.     

Atomic Force Microscopy Analysis of Progenitor Corneal Epithelial Cells Fractionated by a Rapid Centrifugation Isolation Technique

Purpose

To investigate the use of atomic force microscopy (AFM) to image the three groups of corneal epithelial cells fractionated by a novel rapid centrifugation isolation technique.

Methods

Epithelial cells harvested from primary cultures of rabbit limbal rings were centrifuged onto uncoated dishes, first at 1400 rpm and then at 1800 rpm. The adherent cells after centrifugation at 1400 rpm (ATC1), the adherent cells at 1800 rpm (ATC2) and the non-adherent cells at 1800 rpm (NAC) were investigated for BrdU retention and were subjected to contact mode AFM and Transmission Electron Microscopy (TEM).

Results

Compared with unfractionated cells, the ATC1 group, accounting for about 10% of the whole population, was enriched in BrdU label-retaining cells. There were dramatic overall shape, surface membrane and intra-cellular ultrastructure differences noted among ATC1, ATC2 and NAC populations. The whole cell roughness measurements were 21.1±1.5 nm, 79.5±3.4 nm and 103±4.6 nm for the ATC1, ATC2 and NAC groups, respectively. The mero-nucleus roughness measurements were 34.2±1.7 nm, 13.0±0.8 nm and 8.5±0.5 nm in the ATC1, ATC2 and NAC populations, respectively.

Conclusions

AFM was found to be a good tool for distinguishing among the three groups of cells. BrdU label retention, the AFM parameters and TEM together suggest that the ATC1, ATC2 and NAC populations may be progenitor corneal epithelial cells, transit amplifying cells and terminal differentiation cells, respectively.     

Modeling the Soil Water Retention Curves of Soil-Gravel Mixtures with Regression Method on the Loess Plateau of China

Soil water retention parameters are critical to quantify flow and solute transport in vadose zone, while the presence of rock fragments remarkably increases their variability. Therefore a novel method for determining water retention parameters of soil-gravel mixtures is required. The procedure to generate such a model is based firstly on the determination of the quantitative relationship between the content of rock fragments and the effective saturation of soil-gravel mixtures, and then on the integration of this relationship with former analytical equations of water retention curves (WRCs). In order to find such relationships, laboratory experiments were conducted to determine WRCs of soil-gravel mixtures obtained with a clay loam soil mixed with shale clasts or pebbles in three size groups with various gravel contents. Data showed that the effective saturation of the soil-gravel mixtures with the same kind of gravels within one size group had a linear relation with gravel contents, and had a power relation with the bulk density of samples at any pressure head. Revised formulas for water retention properties of the soil-gravel mixtures are proposed to establish the water retention curved surface models of the power-linear functions and power functions. The analysis of the parameters obtained by regression and validation of the empirical models showed that they were acceptable by using either the measured data of separate gravel size group or those of all the three gravel size groups having a large size range. Furthermore, the regression parameters of the curved surfaces for the soil-gravel mixtures with a large range of gravel content could be determined from the water retention data of the soil-gravel mixtures with two representative gravel contents or bulk densities. Such revised water retention models are potentially applicable in regional or large scale field investigations of significantly heterogeneous media, where various gravel sizes and different gravel contents are present.     

Androgen Inhibits Abdominal Fat Accumulation and Negatively Regulates the PCK1 Gene in Male Chickens

Capons are male chickens whose testes have been surgically incised. Capons show a significant increase in fat accumulation compared to intact male chickens. However, while caponization leads to a significant reduction in androgen levels in roosters, little is known about the molecular mechanisms through which androgen status affects lipogenesis in avian species. Therefore, investigation of the influence of androgens on fat accumulation in the chicken will provide insights into this process. In this study, Affymetrix microarray technology was used to analyze the gene expression profiles of livers from capons and intact male chickens because the liver is the major site of lipogenesis in avian species. Through gene ontology, we found that genes involved in hepatic lipogenic biosynthesis were the most highly enriched. Interestingly, among the upregulated genes, the cytosolic form of the phosphoenolpyruvate carboxykinase (PCK1) gene showed the greatest fold change. Additionally, in conjunction with quantitative real-time PCR data, our results suggested that androgen status negatively regulated the PCK1 gene in male chickens.     

Patients with Nipple-Areola Paget’s Disease and Underlying Invasive Breast Carcinoma Have Very Poor Survival: A Matched Cohort Study

Paget’s disease (PD) of the breast is a rare disease. The survival rate of PD was reported to depend on the characteristics of the underlying carcinoma. This study aimed to investigate the characteristics and survival rate of PD patients with underlying invasive breast carcinoma (IBC). Fifty-two patients were diagnosed with PD and an associated IBC from 2001 to 2005 in Fudan University Shanghai Cancer Center. Twenty-four (46.2%) had no clinical manifestation of PD and were diagnosed unexpectedly by a histologic examination. The 52 patients were all recruited in this study as the PD group. They tended to have greater chances of lymph node involvement (53.8% vs. 35.7%), lower hormone receptor expression (34.6% vs. 69.7%), higher human epidermal growth factor receptor 2 (HER2) expression (76.9% vs. 21.3%), and worse survival (5-year relapse-free survival (RFS) 52.2% vs. 86.7%, P<0.01; breast cancer-specific overall survival (OS) 62.1% vs. 91.8%, P<0.01) when compared with patients diagnosed with IBC. A matched study was then performed to investigate whether the poor survival of patients in the PD group was due to the unfavorable prognosis of the underlying IBC. One hundred and fifty-six (3∶1 ratio of controls to PD patients) patients diagnosed with IBC only were recruited into the matched group. The match was conducted according to four variables: dimension of IBC, lymph node status, hormone receptor status and HER2 status. The 5-year RFS (52.2% vs. 81.4%, P<0.01) and OS (62.1% vs. 85.9%, P<0.01) were both lower for patients in the PD group than those in the matched group. Patients with PD and underlying IBC had poor survival. Their survival was worse than that of patients with IBC of similar stage and characteristics. For patients with no clinical PD manifestation who were histologically diagnosed as PD, survival might be worse compared to patients with clinically diagnosed PD.     

Development and Evaluation of a Pseudovirus-Luciferase Assay for Rapid and Quantitative Detection of Neutralizing Antibodies against Enterovirus 71

The level of neutralizing antibodies (NtAb) induced by vaccine inoculation is an important endpoint to evaluate the efficacy of EV71 vaccine. In order to evaluate the efficacy of EV71 vaccine, here, we reported the development of a novel pseudovirus system expression firefly luciferase (PVLA) for the quantitative measurement of NtAb. We first evaluated and validated the sensitivity and specificity of the PVLA method. A total of 326 serum samples from an epidemiological survey and 144 serum specimens from 3 clinical trials of EV71 vaccines were used, and the level of each specimen''s neutralizing antibodies (NtAb) was measured in parallel using both the conventional CPE-based and PVLA-based assay. Against the standard neutralization assay based on the inhibition of the cytopathic effect (CPE), the sensitivity and specificity of the PVLA method are 98% and 96%, respectively. Then, we tested the potential interference of NtAb against hepatitis A virus, Polio-I, Polio-II, and Polio-III standard antisera (WHO) and goat anti-G10/CA16 serum, the PVLA based assay showed no cross-reactivity with NtAb against other specific sera. Importantly, unlike CPE based method, no live replication-competent EV71 is used during the measurement. Taken together, PVLA is a rapid and specific assay with higher sensitivity and accuracy. It could serve as a valuable tool in assessing the efficacy of EV71 vaccines in clinical trials and disease surveillance in epidemiology studies.