Molecular genetic characterization of rice seed lipoxygenase 3 and assessment of its effects on seed longevity

Lipoxygenases (LOXs) are enzymes involved in lipid peroxidation. Here we reported the identification, molecular and functional characterization of the gene encoding rice (Oryza sativa L.) seed LOX3 (sLOX3). Via a map-based cloning strategy we identified Os03g0700400 as the candidate gene encoding sLOX3. Further functional complementary test and biochemical characterization of the recombinant Os03g0700400 protein verified the identification. The sLOX3 gene was highly expressed in roots, moderately in embryos and very weakly in leaves, leaf sheaths and stems. Transient expression experiment (in rice protoplasts) and subsequent laser confocal microscopic analysis demonstrated that the sLOX3 protein was localized into the cytosol. We next showed that overexpression of sLOX3 in a japonica sLOX3-normal rice cultivar, Wuyunjing 7 accelerated the decrease of seed germination ability when the seeds were routinely stored, which demonstrated that sLOX3 had a negative effect on seed longevity (storability). Meanwhile, an increased occurrence of embryo decay was observed in the same transgenic seeds, suggesting that sLOX3 might negatively affect seed longevity by facilitating colonization of particular seed pathogens. Our result forwarded the understanding of the effects of 9-LOX on rice seed longevity.     

hPNAS-4 inhibits proliferation through S phase arrest and apoptosis: underlying action mechanism in ovarian cancer cells

PNAS-4, a novel pro-apoptotic gene, was activated during the early response to DNA damage. Previous studies have shown that hPNAS-4 can inhibit tumor growth when over-expressed in ovarian cancer cells. However, the underlying action mechanism remains elusive. In this work, we found that hPNAS-4 expression was significantly increased in SKOV3 cells when exposed to cisplatin, methyl methanesulfonate or mitomycin C, and that its overexpression could induce proliferation inhibition, S phase arrest and apoptosis in A2780s and SKOV3 ovarian cancer cells. The S phase arrest caused by hPNAS-4 was associated with up-regulation of p21. p21 is p53-dispensable and correlates with activation of ERK, and activation of the Cdc25A-Cdk2-Cyclin E/Cyclin A pathway, while the pro-apoptotic effects of hPNAS-4 were mediated by activation of caspase-9 and -3 other than caspase-8, and accompanied by release of AIF, Smac and cytochrome c into the cytosol. Taken together, these data suggest a new mechanism by which hPNAS-4 inhibits proliferation of ovarian cancer cells by inducing S phase arrest and apoptosis via activation of Cdc25A-Cdk2-Cyclin E/Cyclin A axis and mitochondrial dysfunction-mediated caspase-dependent and -independent apoptotic pathways. To our knowledge, we provide the first molecular evidence for the potential application of hPNAS-4 as a novel target in ovarian cancer gene therapy.     

VRAA: virtualized resource auction and allocation based on incentive and penalty

Virtualization is widely used in cloud computing environments to efficiently manage resources, but it also raises several challenges. One of them is the fairness issue of resource allocation among virtual machines. Traditional virtualized resource allocation approaches distribute physical resources equally without taking into account the actual workload of each virtual machine and thus often leads to wasting. In this paper, we propose a virtualized resource auction and allocation model (VRAA) based on incentive and penalty to correct this wasting problem. In our approach, we use Nash equilibrium of cooperative games to fairly allocate resources among multiple virtual machines to maximize revenue of the system. To illustrate the effectiveness of the proposed approach, we then apply the basic laws of auction gaming to investigate how CPU allocation and contention can affect applications’ performance (i.e., response time), and its effect on CPU utilization. We find that in our VRAA model, the fairness index is high, and the resource allocation is closely proportional to the actual workloads of the virtual machines, so the wasting of resources is reduced. Experiment results show that our model is general, and can be applied to other virtualized non-CPU resources.     

ORTHRUS: a lightweighted block-level cloud storage system

Taking advantage of distributed storage technology and virtualization technology, cloud storage systems provide virtual machine clients customizable storage service. They can be divided into two types: distributed file system and block level storage system. There are two disadvantages in existing block level storage system: Firstly, Some of them are tightly coupled with their cloud computing environments. As a result, it’s hard to extend them to support other cloud computing platforms; Secondly, The bottleneck of volume server seriously affects the performance and reliability of the whole system. In this paper we present a lightweighted block-level storage system for clouds—ORTHRUS, based on virtualization technology. We first design the architecture with multiple volume servers and its workflows, which can improve system performance and avoid the problem. Secondly, we propose a Listen-Detect-Switch mechanism for ORTHRUS to deal with contingent volume servers’ failure. At last we design a strategy that dynamically balances load between multiple volume servers. We characterize machine capability and load quantity with black box model, and implement the dynamic load balance strategy which is based on genetic algorithm. Extensive experimental results show that the aggregated I/O throughputs of ORTHRUS are significantly improved (approximately two times of that in Orthrus), and both I/O throughputs and IOPS are also remarkably improved (about 1.8 and 1.2 times, respectively) by our dynamic load balance strategy.     

Effect of the primary host for production of both sexes on the mating interaction in an autoparasitoid species

Encarsia sophia (Girault and Dodd) is an autoparasitoid in the hymenopteran family Aphelinidae. The females develop as primary parasitoids on whitefly nymphs (primary hosts), whereas the males develop as hyperparasitoids on their own species or on other primary parasitoid species (secondary hosts). The autoparasitoids not only parasitise whiteflies but also kill them with strong host-feeding capacity. In this study, female and male E. sophia were reared on the primary hosts Trialeurodes vaporariorum and Bemisia tabaci ‘Q’, and the host-feeding and parasitism of wasps on both whitefly species were determined for the four possible different mating combinations: (i) E. sophia females reared on B. tabaci (ESF-BT) mated with E. sophia males from B. tabaci (ESM-BT), (ii) E. sophia females reared on T. vaporariorum (ESF-TV) mated with E. sophia males from T. vaporariorum (ESM-TV), (iii) ESF-BT mated with ESM-TV, and (iv) ESF-TV mated with ESM-BT. ESF-TV mated with ESM-TV killed the largest percentage of whitefly nymphs through host feeding. The ESF-TV with larger body size mating with larger ESM-TV killed more whitefly nymphs through host feeding than those mating with smaller ESM-BT. Whether B. tabaci or T. vaporariorum were used as hosts, ESF-TV mated with ESM-TV and ESM-BT and ESF-BT mated with ESM-BT significantly parasitised more whitefly nymphs than ESF-BT mated with ESM-TV. In general, ESF-BT mated with ESM-TV killed significantly fewer whitefly nymphs through parasitism and host feeding than the other three mating combinations on both whitefly species. These results indicated that the performance of autoparasitoids on insect pests was not only dependent on females but was also affected by mating with males from different primary host species.     

Construction of a genomewide RNAi mutant library in rice

Long hairpin RNA (hpRNA) transgenes are a powerful tool for gene function studies in plants, but a genomewide RNAi mutant library using hpRNA transgenes has not been reported for plants. Here, we report the construction of a hpRNA library for the genomewide identification of gene function in rice using an improved rolling circle amplification‐mediated hpRNA (RMHR) method. Transformation of rice with the library resulted in thousands of transgenic lines containing hpRNAs targeting genes of various function. The target mRNA was down‐regulated in the hpRNA lines, and this was correlated with the accumulation of siRNAs corresponding to the double‐stranded arms of the hpRNA. Multiple members of a gene family were simultaneously silenced by hpRNAs derived from a single member, but the degree of such cross‐silencing depended on the level of sequence homology between the members as well as the abundance of matching siRNAs. The silencing of key genes tended to cause a severe phenotype, but these transgenic lines usually survived in the field long enough for phenotypic and molecular analyses to be conducted. Deep sequencing analysis of small RNAs showed that the hpRNA‐derived siRNAs were characteristic of Argonaute‐binding small RNAs. Our results indicate that RNAi mutant library is a high‐efficient approach for genomewide gene identification in plants.     

Proteomic analysis of kidneys from selenoprotein M transgenic rats in response to increased bioability of selenium

Background

To characterize changes in global protein expression in kidneys of transgenic rats overexpressing human selenoprotein M (SelM) in response to increased bioabivility of selenium (Sel), total proteins extracted from kidneys of 10-week-old CMV/hSelM Tg and wild-type rats were separated by 2-dimensional gel electrophoresis and measured for changes in expression.

Results

Ten and three proteins showing high antioxidant enzymatic activity were up- and down-regulated, respectively, in SelM-overexpressing CMV/hSelM Tg rats compared to controls based on an arbitrary 2-fold difference. Up-regulated proteins included LAP3, BAIAP2L1, CRP2, CD73 antigen, PDGF D, KIAA143 homolog, PRPPS-AP2, ZFP313, HSP-60, and N-WASP, whereas down-regulated proteins included ALKDH3, rMCP-3, and STC-1. After Sel treatment, five of the up-regulated proteins were significantly increased in expression in wild-type rats, whereas there were no changes in CMV/hSelM Tg rats. Only two of the down-regulated proteins showed reduced expression in wild-type and Tg rats after Sel treatment.

Conclusions

These results show the primary novel biological evidences that new functional protein groups and individual proteins in kidneys of Tg rats relate to Sel biology including the response to Sel treatment and SelM expression.     

Generation of Five Human Lactoferrin Transgenic Cloned Goats Using Fibroblast Cells and Their Methylation Status of Putative Differential Methylation Regions of IGF2R and H19 Imprinted Genes

Background

Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos. According to the studies on cattle and mice, DNA methylation of some imprinted genes, which plays a vital role in the reprogramming of embryo in NT maybe an underlying mechanism.

Methodology/Principal Findings

Fibroblast cells were derived from the ear of a two-month-old goat. The vector expressing hLF was constructed and transfected into fibroblasts. G418 selection, EGFP expression, PCR, and cell cycle distribution were applied sequentially to select transgenic cells clones. After NT and embryo transfer, five transgenic cloned goats were obtained from 240 cloned transgenic embryos. These transgenic goats were identified by 8 microsatellites genotyping and southern blot. Of the five transgenic goats, 3 were lived after birth, while 2 were dead during gestation. We compared differential methylation regions (DMR) pattern of two paternally imprinted genes (H19 and IGF2R) of the ear tissues from the lived transgenic goats, dead transgenic goats, and control goats from natural reproduction. Hyper-methylation pattern appeared in cloned aborted goats, while methylation status was relatively normal in cloned lived goats compared with normal goats.

Conclusions/Significance

In this study, we generated five hLF transgenic cloned goats by SCNT. This is the first time the DNA methylation of lived and dead transgenic cloned goats was compared. The results demonstrated that the methylation status of DMRs of H19 and IGF2R were different in lived and dead transgenic goats and therefore this may be potentially used to assess the reprogramming status of transgenic cloned goats. Understanding the pattern of gene imprinting may be useful to improve cloning techniques in future.     

Insulin Resistance Is an Independent Determinate of ED in Young Adult Men

Background

Insulin resistance (IR) triggers endothelial dysfunction, which contributes to erectile dysfunction (ED) and cardiovascular disease.

Aim

To evaluate whether IR was related to ED in young adult patients.

Methods

A total of 283 consecutive men complaining of ED at least six months were enrolled, with a full medical history, physical examination, and laboratory tests collected. Quantitative Insulin Sensitivity Check Index (QUICKI) was used to determine IR. The severity of ED was assessed by IIEF-5 questionnaire. Endothelial function was assessed by ultrasonographic examination of brachial artery flow mediated dilation (FMD).

Results

IR was detected in 52% patients. Subjects with IR had significant higher total cholesterol, triglycerides, low density lipoprotein-cholesterol (LDL-c), glycated haemoglobin (HBA1c), high sensitivity C-reactive protein (hs-CRP) and body mass index (BMI), but showed significant lower IIEF-5 score, FMD%, high density lipoprotein -cholesterol (HDL-c), testosterone, sex hormone binding globulin (SHBG) levels than patients without IR. Multiple regression analysis showed QUICKI and testosterone were independent predictors of IIEF-5 score. Furthermore, the incidence of IR was correlated with the severity of ED.

Conclusions

Compared with other CVFs, IR was found as the most prevalent in our subjects. Besides, IR was independently associated with ED and its severity, suggesting an adverse effect of insulin resistance on erectile function.