Identification of Pns12 as the second silencing suppressor of <Emphasis Type="Italic">Rice gall dwarf virus</Emphasis>

RNA silencing is a conserved mechanism found ubiquitously in eukaryotic organisms. It has been used to regulate gene expression and development. In addition, RNA silencing serves as an important mechanism in plants’ defense against invasive nucleic acids, such as viruses, transposons, and transgenes. As a counter-defense, most plants, and some animal viruses, encode RNA silencing suppressors to interfere at one or several points of the silencing pathway. In this study, we showed that Pns12 of RGDV (Rice gall dwarf virus) exhibits silencing suppressor activity on the reporter green fluorescent protein in transgenic Nicotiana benthamiana line 16c. Pns12 of RGDV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA. Expression of Pns12 also enhanced Potato virus X pathogenicity in N. benthamiana. Collectively, these results suggested that RGDV Pns12 functions as a virus suppressor of RNA silencing, which might target an upstream step of dsRNA formation in the RNA silencing pathway. Furthermore, we showed that Pns12 is localized mainly in the nucleus of N. benthamiana leaf cells.     

Mapping, expression and regulation of the TRα gene in porcine adipose tissue

Thyroid hormone receptors (TR) are members of the nuclear receptor superfamily. There are at least two TR isoforms, TRα and TRβ. The TRα isoform plays a critical role in mediating the action of thyroid hormone in adipose tissue. We mapped the porcine TRα gene to chromosome 12 p11-p13, by using the ImpRH panel. We examined tissue-localization of TRα and determined expression patterns of TRα in porcine adipose tissue with quantitative real-time PCR. TRα was expressed in all tissues, including heart, liver, spleen, stomach, pancreas, brain, small intestine, skeletal muscle, and subcutaneous adipose tissue. In the adipose tissue, the expression of TRα decreased postnatally. Compared to Yorkshire pigs, Jinhua pigs had significantly lower expression levels of TRα gene in the subcutaneous fat tissue. The expression levels of β2-AR, HSL and ATGL were also significantly lower in Jinhua pigs than in Yorkshire pigs. However, no significant differences in PPARγ and SREBP-1C expression levels were found between Jinhua and Yorkshire pigs. Incubation of porcine adipose tissue explants with high doses of isoproterenol (100 and 1000 nM) significantly increased the expression levels of TRα. We conclude that there is considerable evidence that TRα plays an important role in fat deposition in porcine adipose tissue.     

Quantitative proteomics analysis of phosphorylated proteins in the hippocampus of Alzheimer's disease subjects

Phosphorylation on tyrosine, threonine and serine residues represents one of the most important post-translational modifications and is a key regulator of cellular signaling of multiple biological processes that require a strict control by protein kinases and protein phosphatases. Abnormal protein phosphorylation has been associated with several human diseases including Alzheimer's disease (AD). One of the characteristic hallmarks of AD is the presence of neurofibrillary tangles, composed of microtubule-associated, abnormally hyperphosphorylated tau protein. However, several others proteins showed altered phosphorylation levels in AD suggesting that deregulated phosphorylation may contribute to AD pathogenesis. Phosphoproteomics has recently gained attention as a valuable approach to analyze protein phosphorylation, both in a quantitative and a qualitative way. We used the fluorescent phosphospecific Pro-Q Diamond dye to identify proteins that showed alterations in their overall phosphorylation in the hippocampus of AD vs. control (CTR) subjects. Significant changes were found for 17 proteins involved in crucial neuronal process such as energy metabolism or signal transduction. These phosphoproteome data may provide new clues to better understand molecular pathways that are deregulated in the pathogenesis and progression of AD.     

Phase I randomised clinical trial of an HIV-1(CN54), clade C, trimeric envelope vaccine candidate delivered vaginally

We conducted a phase 1 double-blind randomised controlled trial (RCT) of a HIV-1 envelope protein (CN54 gp140) candidate vaccine delivered vaginally to assess immunogenicity and safety. It was hypothesised that repeated delivery of gp140 may facilitate antigen uptake and presentation at this mucosal surface. Twenty two healthy female volunteers aged 18-45 years were entered into the trial, the first receiving open-label active product. Subsequently, 16 women were randomised to receive 9 doses of 100 μg of gp140 in 3 ml of a Carbopol 974P based gel, 5 were randomised to placebo solution in the same gel, delivered vaginally via an applicator. Participants delivered the vaccine three times a week over three weeks during one menstrual cycle, and were followed up for two further months. There were no serious adverse events, and the vaccine was well tolerated. No sustained systemic or local IgG, IgA, or T cell responses to the gp140 were detected following vaginal immunisations. Repeated vaginal immunisation with a HIV-1 envelope protein alone formulated in Carbopol gel was safe, but did not induce local or systemic immune responses in healthy women. TRIAL REGISTRATION: ClinicalTrials.gov NCT00637962.     

Classification and analysis of regulatory pathways using graph property, biochemical and physicochemical property, and functional property

Given a regulatory pathway system consisting of a set of proteins, can we predict which pathway class it belongs to? Such a problem is closely related to the biological function of the pathway in cells and hence is quite fundamental and essential in systems biology and proteomics. This is also an extremely difficult and challenging problem due to its complexity. To address this problem, a novel approach was developed that can be used to predict query pathways among the following six functional categories: (i) “Metabolism”, (ii) “Genetic Information Processing”, (iii) “Environmental Information Processing”, (iv) “Cellular Processes”, (v) “Organismal Systems”, and (vi) “Human Diseases”. The prediction method was established trough the following procedures: (i) according to the general form of pseudo amino acid composition (PseAAC), each of the pathways concerned is formulated as a 5570-D (dimensional) vector; (ii) each of components in the 5570-D vector was derived by a series of feature extractions from the pathway system according to its graphic property, biochemical and physicochemical property, as well as functional property; (iii) the minimum redundancy maximum relevance (mRMR) method was adopted to operate the prediction. A cross-validation by the jackknife test on a benchmark dataset consisting of 146 regulatory pathways indicated that an overall success rate of 78.8% was achieved by our method in identifying query pathways among the above six classes, indicating the outcome is quite promising and encouraging. To the best of our knowledge, the current study represents the first effort in attempting to identity the type of a pathway system or its biological function. It is anticipated that our report may stimulate a series of follow-up investigations in this new and challenging area.     

PIPKIγ regulates focal adhesion dynamics and colon cancer cell invasion

Focal adhesion assembly and disassembly are essential for cell migration and cancer invasion, but the detailed molecular mechanisms regulating these processes remain to be elucidated. Phosphatidylinositol phosphate kinase type Iγ (PIPKIγ) binds talin and is required for focal adhesion formation in EGF-stimulated cells, but its role in regulating focal adhesion dynamics and cancer invasion is poorly understood. We show here that overexpression of PIPKIγ promoted focal adhesion formation, whereas cells expressing either PIPKIγ^K188,200R or PIPKIγ^D316K, two kinase-dead mutants, had much fewer focal adhesions than those expressing WT PIPKIγ in CHO-K1 cells and HCT116 colon cancer cells. Furthermore, overexpression of PIPKIγ, but not PIPKIγ^K188,200R, resulted in an increase in both focal adhesion assembly and disassembly rates. Depletion of PIPKIγ by using shRNA strongly inhibited formation of focal adhesions in HCT116 cells. Overexpression of PIPKIγ^K188,200R or depletion of PIPKIγ reduced the strength of HCT116 cell adhesion to fibronection and inhibited the invasive capacities of HCT116 cells. PIPKIγ depletion reduced PIP₂ levels to ∼40% of control and PIP₃ to undetectable levels, and inhibited vinculin localizing to focal adhesions. Taken together, PIPKIγ positively regulates focal adhesion dynamics and cancer invasion, most probably through PIP₂-mediated vinculin activation.     

Predicting protein phenotypes based on protein-protein interaction network

Background

Identifying associated phenotypes of proteins is a challenge of the modern genetics since the multifactorial trait often results from contributions of many proteins. Besides the high-through phenotype assays, the computational methods are alternative ways to identify the phenotypes of proteins.

Methodology/Principal Findings

Here, we proposed a new method for predicting protein phenotypes in yeast based on protein-protein interaction network. Instead of only the most likely phenotype, a series of possible phenotypes for the query protein were generated and ranked acording to the tethering potential score. As a result, the first order prediction accuracy of our method achieved 65.4% evaluated by Jackknife test of 1,267 proteins in budding yeast, much higher than the success rate (15.4%) of a random guess. And the likelihood of the first 3 predicted phenotypes including all the real phenotypes of the proteins was 70.6%.

Conclusions/Significance

The candidate phenotypes predicted by our method provided useful clues for the further validation. In addition, the method can be easily applied to the prediction of protein associated phenotypes in other organisms.