Anthony Kwan Leung

Directed Proteomic Analysis of the Human Nucleolus

BACKGROUND The nucleolus is a subnuclear organelle containing the ribosomal RNA gene clusters and ribosome biogenesis factors. Recent studies suggest it may also have roles in RNA transport, RNA modification, and cell cycle regulation. Despite over 150 years of research into nucleoli, many aspects of their structure and function remain uncharacterized. RESULTS We report a proteomic analysis of human nucleoli. Using a combination of mass spectrometry (MS) and sequence database searches, including online analysis of the draft human genome sequence, 271 proteins were identified. Over 30% of the nucleolar proteins were encoded by novel or uncharacterized genes, while the known proteins included several unexpected factors with no previously known nucleolar functions. MS analysis of nucleoli isolated from HeLa cells in which transcription had been inhibited showed that a subset of proteins was enriched. These data highlight the dynamic nature of the nucleolar proteome and show that proteins can either associate with nucleoli transiently or accumulate only under specific metabolic conditions. CONCLUSIONS This extensive proteomic analysis shows that nucleoli have a surprisingly large protein complexity. The many novel factors and separate classes of proteins identified support the view that the nucleolus may perform additional functions beyond its known role in ribosome subunit biogenesis. The data also show that the protein composition of nucleoli is not static and can alter significantly in response to the metabolic state of the cell.

Paraspeckles : a novel nuclear domain

BACKGROUND The cell nucleus contains distinct classes of subnuclear bodies, including nucleoli, splicing speckles, Cajal bodies, gems, and PML bodies. Many nuclear proteins are known to interact dynamically with one or other of these bodies, and disruption of the specific organization of nuclear proteins can result in defects in cell functions and may cause molecular disease. RESULTS A proteomic study of purified human nucleoli has identified novel proteins, including Paraspeckle Protein 1 (PSP1) (see accompanying article, this issue of Current Biology). Here we show that PSP1 accumulates in a new nucleoplasmic compartment, termed paraspeckles, that also contains at least two other protein components: PSP2 and p54/nrb. A similar pattern of typically 10 to 20 paraspeckles was detected in all human cell types analyzed, including primary and transformed cells. Paraspeckles correspond to discrete bodies in the interchromatin nucleoplasmic space that are often located adjacent to splicing speckles. A stable cell line expressing YFP-PSP1 has been established and used to demonstrate that PSP1 interacts dynamically with nucleoli and paraspeckles in living cells. The three paraspeckle proteins relocalize quantitatively to unique cap structures at the nucleolar periphery when transcription is inhibited. CONCLUSIONS We have identified a novel nuclear compartment, termed paraspeckles, found in both primary and transformed human cells. Paraspeckles contain at least three RNA binding proteins that all interact dynamically with the nucleolus in a transcription-dependent fashion.

Condensed mitotic chromatin is accessible to transcription factors and chromatin structural proteins

During mitosis, chromosomes are highly condensed and transcription is silenced globally. One explanation for transcriptional repression is the reduced accessibility of transcription factors. To directly test this hypothesis and to investigate the dynamics of mitotic chromatin, we evaluate the exchange kinetics of several RNA polymerase I transcription factors and nucleosome components on mitotic chromatin in living cells. We demonstrate that these factors rapidly exchange on and off ribosomal DNA clusters and that the kinetics of exchange varies at different phases of mitosis. In addition, the nucleosome component H1c-GFP also shows phase-specific exchange rates with mitotic chromatin. Furthermore, core histone components exchange at detectable levels that are elevated during anaphase and telophase, temporally correlating with H3-K9 acetylation and recruitment of RNA polymerase II before the onset of bulk RNA synthesis at mitotic exit. Our findings indicate that mitotic chromosomes in general and ribosomal genes in particular, although highly condensed, are accessible to transcription factors and chromatin proteins. The phase-specific exchanges of nucleosome components during late mitotic phases are consistent with an emerging model of replication independent core histone replacement.

In vivo analysis of NHPX reveals a novel nucleolar localization pathway involving a transient accumulation in splicing speckles

The NHPX protein is a nucleolar factor that binds directly to a conserved RNA target sequence found in nucleolar box C/D snoRNAs and in U4 snRNA. Using enhanced yellow fluorescent protein (EYFP)– and enhanced cyan fluorescent protein–NHPX fusions, we show here that NHPX is specifically accumulated in both nucleoli and Cajal bodies (CBs) in vivo. The fusion proteins display identical localization patterns and RNA binding specificities to the endogenous NHPX. Analysis of a HeLa cell line stably expressing EYFP–NHPX showed that the nucleolar accumulation of NHPX was preceded by its transient accumulation in splicing speckles. Only newly expressed NHPX accumulated in speckles, and the nucleolar pool of NHPX did not interchange with the pool in speckles, consistent with a unidirectional pathway. The transient accumulation of NHPX in speckles prior to nucleoli was observed in multiple cell lines, including primary cells that lack CBs. Inhibitor studies indicated that progression of newly expressed NHPX from speckles to nucleoli was dependent on RNA polymerase II transcription, but not on RNA polymerase I activity. The data show a specific temporal pathway involving the sequential and directed accumulation of NHPX in distinct subnuclear compartments, and define a novel mechanism for nucleolar localization.

A Novel Root System for Simulating Transpiration-Induced Soil Suction in Centrifuge

Plant transpiration has potential beneficial effects to slope stability because it increases soil suction, which results in not only an increase in shear strength but also a decrease in water permeability. Although the effects of vegetation on slope stability have been recently investigated using centrifuge, contributions of plant-induced suction are ignored. In order to simulate transpiration-induced suction in a centrifuge model test, a novel root system that enables suction to be induced and controlled at high-g are developed and verified in this study. This new root system consists of a high air-entry value (AEV) porous filter, cellulose acetate, which has scaled mechanical properties, including tensile strength, elastic modulus, and axial rigidity, similar to living roots. This filter is fully saturated with de-aired water and it is connected to an airtight chamber for controlling vacuum pressures. The function of the water-saturated porous filter is to maintain hydraulic gradient between soil and the root system for any vacuum pressure lower than the AEV of the filter. Any reduction of soil moisture due to applied vacuum hence induces suction. Suctions induced by the new root system were verified to be consistent with those induced by a living tree (Schefflera heptaphylla) at 1-g and that retained by vegetation in the field. Both vertical and horizontal influence zones of suction of the living tree were captured. For centrifuge tests carried out at 15-g, suctions of up to 25 kPa can be simulated.

Correlating hydrologic reinforcement of vegetated soil with plant traits during establishment of woody perennials

Background and aimsVegetation stabilizes slopes via root mechanical reinforcement and hydrologic reinforcement induced by transpiration. Most studies have focused on mechanical reinforcement and its correlation with plant biomechanical traits. The correlations however generally ignore the effects of hydrologic reinforcement. This study aims to quantify the hydrologic reinforcement associated with ten woody species and identify correlations with relevant plant traits.MethodsTen species widespread in Europe, which belong to Aquifoliaceae, Betulaceae, Buxaceae, Celastraceae, Fabaceae, Oleaceae and Salicaceae families, were planted in pots of sandy loam soil. Each planted pot was irrigated and then left to transpire. Soil strength, matric suction and plant traits were measured.ResultsTranspiration-induced suction was linearly correlated with soil penetration resistance for the ten species due to their different transpiration rates i.e. both suction and soil penetration resistance induced by Hazel and Blackthorn (deciduous) were five times greater than those by Holly and European Box (evergreens). Specific leaf area and root length density correlated with hydrologic reinforcement. The root:shoot ratio correlated best with the hydrologic reinforcement.ConclusionsSpecific leaf area, root length density and root:shoot ratio explained the tenfold differences in hydrologic reinforcement provided by the ten different species.

Effects of Plant Transpiration on Suction Distribution in a Vegetated Soil Slope

Extensive studies have been carried out to investigate the use of plant roots as a mechanical reinforcement for slope stabilisation. However, although plant is well-recognised to induce soil suction via transpiration, any of its contribution to slope stability is rarely investigated. The objective of this study is to investigate the plant-induced suction distribution on a slope vegetated with multiple plants. A series of parametric study is conducted to investigate the effects of plant number and spacing on induced suction and its influence zone. In each analysis, the ground is subjected to 6-month drying under a constant potential transpiration rate. Any computed variations of plant-induced suction influence zone in each case are discussed.

Pilot trial study of a compact macro-filtration membrane bioreactor process for saline wastewater treatment

Conventional membrane bioreactor (MBR) systems have increasingly been studied in recent decades. However, their applications have been limited due to their drawbacks such as low flux, membrane fouling, and high operating cost. In this study, a compact macro-filtration MBR (MfMBR) process was developed by using a large pore size membrane to mitigate the membrane fouling problem. A pilot trial of MfMBR process was set up and operated to treat 10 m(3)/day of saline wastewater within 4 h. The system was operated under an average permeate flux of 13.1 m(3)/(m(2)·day) for 74 days. The average total suspended solids, total chemical oxygen demand, biological oxygen demand, total Kjeldahl nitrogen, and total nitrogen removal efficiencies achieved were 94.3, 83.1, 98.0, 93.1, and 63.3%, respectively, during steady-state operation. The confocal laser scanning microscopy image indicated that the backwash could effectively remove the bio-cake and dead bacteria. Thus, the results showed that the MfMBR process, which is essentially a primary wastewater treatment process, had the potential to yield the same high quality effluent standards as the secondary treatment process; thereby suggesting that it could be used as an option when the economic budget and/or land space is limited.

Numerical modelling of methane oxidation efficiency and coupled water-gas-heat reactive transfer in a sloping landfill cover

Microbial aerobic methane oxidation in unsaturated landfill cover involves coupled water, gas and heat reactive transfer. The coupled process is complex and its influence on methane oxidation efficiency is not clear, especially in steep covers where spatial variations of water, gas and heat are significant. In this study, two-dimensional finite element numerical simulations were carried out to evaluate the performance of unsaturated sloping cover. The numerical model was calibrated using a set of flume model test data, and was then subsequently used for parametric study. A new method that considers transient changes of methane concentration during the estimation of the methane oxidation efficiency was proposed and compared against existing methods. It was found that a steeper cover had a lower oxidation efficiency due to enhanced downslope water flow, during which desaturation of soil promoted gas transport and hence landfill gas emission. This effect was magnified as the cover angle and landfill gas generation rate at the bottom of the cover increased. Assuming the steady-state methane concentration in a cover would result in a non-conservative overestimation of oxidation efficiency, especially when a steep cover was subjected to rainfall infiltration. By considering the transient methane concentration, the newly-modified method can give a more accurate oxidation efficiency.

Theoretical analysis of coupled effects of microbe and root architecture on methane oxidation in vegetated landfill covers

Reduction of soil moisture by plant root-water uptake could improve soil aeration for microbial aerobic methane oxidation (MAMO) in a landfill cover, but excessive soil moisture removal could suppress microbial activity due to water shortage. Existing models ignore the coupled microbe-vegetation interaction. It is thus not known whether the presence of plants is beneficial or adverse to MAMO. This study proposes a newly-improved theoretical model that couples the effects of root-water uptake and microbial activity for capturing water-gas flow and MAMO in unsaturated soils. Parametric studies are conducted to investigate the effects of root characteristics and transpiration rate on MAMO efficiency. Uniform, parabolic, exponential and triangular root architectures are considered. Ignoring the effects of water shortage on microbe over-predicts the MAMO efficiency significantly, especially for plants with traits that give high root-water uptake ability (i.e., uniformly-rooted and long root length). The effects of plants on MAMO efficiency depends on the initial soil moisture strongly. If the soil is too dry (i.e., close to the permanent wilting point), plant-water uptake, with any root architecture considered, would reduce MAMO efficiency as further soil water removal by plants suppresses microbial activity. Plants with exponential or triangular root architectures could preserve 10% higher MAMO than the other two cases. These two architectures are more capable of minimizing the adverse effects of root-water uptake due to microbial water shortage. This implies that high-water-demand plants such as those with long root length and with uniform or parabolic root architectures require more frequent irrigation to prevent from excessive reduction of MAMO efficiency.