Supramolecular structure in the membrane of Staphylococcus aureus

The fundamental processes of life are organized and based on common basic principles. Molecular organizers, often interacting with the membrane, capitalize on cellular polarity to precisely orientate essential processes. The study of organisms lacking apparent polarity or known cellular organizers (e.g., the bacterium Staphylococcus aureus) may enable the elucidation of the primal organizational drive in biology. How does a cell choose from infinite locations in its membrane? We have discovered a structure in the S. aureus membrane that organizes processes indispensable for life and can arise spontaneously from the geometric constraints of protein complexes on membranes. Building on this finding, the most basic cellular positioning system to optimize biological processes, known molecular coordinators could introduce further levels of complexity. All life demands the temporal and spatial control of essential biological functions. In bacteria, the recent discovery of coordinating elements provides a framework to begin to explain cell growth and division. Here we present the discovery of a supramolecular structure in the membrane of the coccal bacterium Staphylococcus aureus, which leads to the formation of a large-scale pattern across the entire cell body; this has been unveiled by studying the distribution of essential proteins involved in lipid metabolism (PlsY and CdsA). The organization is found to require MreD, which determines morphology in rod-shaped cells. The distribution of protein complexes can be explained as a spontaneous pattern formation arising from the competition between the energy cost of bending that they impose on the membrane, their entropy of mixing, and the geometric constraints in the system. Our results provide evidence for the existence of a self-organized and nonpercolating molecular scaffold involving MreD as an organizer for optimal cell function and growth based on the intrinsic self-assembling properties of biological molecules

Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

Aspartate residues function as proton acceptors in catalysis and are involved in ionic interactions stabilizing subunit assembly. In an attempt to unravel the role of a conserved aspartate (D89) in sheep-liver tetrameric serine hydroxymethyltransferase (SHMT), it was converted into aspargine by site-directed mutagenesis. The purified D89N mutant enzyme had a lower specific activity compared with the wild-type enzyme. It was a mixture of dimers and tetramers with the proportion of tetramers increasing with an increase in the pyridoxal-5′-phosphate (PLP) concentration used during purification. The D89N mutant tetramer was as active as the wild-type enzyme and had similar kinetic and spectral properties in the presence of 500 μM PLP. The quinonoid spectral intermediate commonly seen in the case of SHMT was also seen in the case of D89N mutant tetramer, although the amount of intermediate formed was lower. Although the purified dimer exhibited visible absorbance at 425 nm, it had a negligible visible CD spectrum at 425 nm and was only 5% active. The apo-D89N mutant tetramer was a dimer unlike the apo-form of the wild-type enzyme which was present predominantly as a tetramer. Furthermore the apo mutant dimer could not be reconstituted to the holo-form by the addition of excess PLP, suggesting that dimer-dimer interactions are weak in this mutant. The recently published crystal structure of human liver cytosolic recombinant SHMT indicates that this residue (D90 in the human enzyme) is located at the N-terminal end of the fourth helix of one subunit and packs against K39 from the second N-terminal helix of the other symmetry related subunit forming the tight dimer. D89 is at the interface of tight dimers where the PLP 5'-phosphate is also bound. Mutation of D89 could lead to weakened ionic interactions in the tight dimer interface, resulting in decreased affinity of the enzyme for the cofactor

Measurement and simulation of one-dimensional transient three phase flow for monotonic liquid drainage

Simultaneous movement of oil, water, and air in a sandy porous medium was investigated experimentally under transient flow conditions and results were compared to numerical simulations employing a finite element multiphase flow code. The liquid hydrocarbon was Soltrol 170, a low-density branched alkane mixture. Liquid saturations were measured using a collinear dual-energy gamma radiation apparatus and liquid pressures were measured using hydrophilic (untreated) and hydrophobic (treated) ceramic tensiometers connected to pressure transducers. The experimental regime was selected to impose monotonically draining water and total liquid saturation paths to avoid hysteretic effects. Measured saturations and pressures are compared to values obtained from numerical simulations of the experiment using a finite element solution of the governing multiphase flow equations assuming negligible gas pressure gradients. Functional relationships between permeabilitiesk, saturations S, and capillary pressures P employed in the numerical model were estimated by two calibration methods which require different degrees of experimental effort. Measured transient water saturation versus oil-water capillary head data agreed well with predictions from static air-water S-P relations and interfacial tension data. Transient total liquid saturation versus air-oil capillary head data deviated more severely from the scaled air-water S-P data, possibly reflecting noncompliance with the assumption of negligible gas pressure gradients. Reasonably good agreement was observed between measured and numerically simulated water and oil saturations and pressures in space and time. Sensitivity of the numerical results to calibration method was not great

Improving signal-to-noise ratio of structured light microscopy based on photon reassignment

In this paper, we report a method for 3D visualization of a biological specimen utilizing a structured light wide-field microscopic imaging system. This method improves on existing structured light imaging modalities by reassigning fluorescence photons generated from off-focal plane excitation, improving in-focus signal strength. Utilizing a maximum likelihood approach, we identify the most likely fluorophore distribution in 3D that will produce the observed image stacks under structured and uniform illumination using an iterative maximization algorithm. Our results show the optical sectioning capability of tissue specimens while mostly preserving image stack photon count, which is usually not achievable with other existing structured light imaging methods

Sub-population analysis based on temporal features of high content images

Background: High content screening techniques are increasingly used to understand the regulation and progression of cell motility. The demand of new platforms, coupled with availability of terabytes of data has challenged the traditional technique of identifying cell populations by manual methods and resulted in development of high-dimensional analytical methods. Results: In this paper, we present sub-populations analysis of cells at the tissue level by using dynamic features of the cells. We used active contour without edges for segmentation of cells, which preserves the cell morphology, and autoregressive modeling to model cell trajectories. The sub-populations were obtained by clustering static, dynamic and a combination of both features. We were able to identify three unique sub-populations in combined clustering. Conclusion: We report a novel method to identify sub-populations using kinetic features and demonstrate that these features improve sub-population analysis at the tissue level. These advances will facilitate the application of high content screening data analysis to new and complex biological problems.Computation and Systems Biology Programme of Singapore--Massachusetts Institute of Technology Allianc

Underground Wireless Channel Bandwidth and Capacity

The UG channel bandwidth and capacity are vital parameters in wireless underground communication system design. In this chapter, a comprehensive analysis of the wireless underground channel capacity is presented. The impact of soil on return loss, bandwidth, and path loss is discussed. The results of underground multi-carrier modulation capacity are also outlined. Moreover, the single user capacity and multi-carrier capacity are also introduced with an in-depth treatment of soil texture, soil moisture, and distance effects on channel capacity. Finally, the chapter is concluded with a discussion of challenges and open research issues

Impacts of the 2004 tsunami on groundwater resources in Sri Lanka, Water Resour

[1] The 26 December 2004 tsunami caused widespread destruction and contamination of coastal aquifers across southern Asia. Seawater filled domestic open dug wells and also entered the aquifers via direct infiltration during the first flooding waves and later as ponded seawater infiltrated through the permeable sands that are typical of coastal aquifers. In Sri Lanka alone, it is estimated that over 40,000 drinking water wells were either destroyed or contaminated. From February through September 2005, a team of United States, Sri Lankan, and Danish water resource scientists and engineers surveyed the coastal groundwater resources of Sri Lanka to develop an understanding of the impacts of the tsunami and to provide recommendations for the future of coastal water resources in south Asia. In the tsunami-affected areas, seawater was found to have infiltrated and mixed with fresh groundwater lenses as indicated by the elevated groundwater salinity levels. Seawater infiltrated through the shallow vadose zone as well as entered aquifers directly through flooded open wells. Our preliminary transport analysis demonstrates that the intruded seawater has vertically mixed in the aquifers because of both forced and free convection. Widespread pumping of wells to remove seawater was effective in some areas, but overpumping has led to upconing of the saltwater interface and rising salinity. We estimate that groundwater recharge from several monsoon seasons will reduce salinity of many sandy Sri Lankan coastal aquifers. However, the continued sustainability of these small and fragile aquifers for potable water will be difficult because of the rapid growth of human activities that results in more intensive groundwater pumping and increased pollution. Long-term sustainability of coastal aquifers is also impacted by the decrease in sand replenishment of the beaches due to sand mining and erosion

Amplification and demultiplexing in insulin-regulated Akt protein kinase pathway in adipocytes.

Akt plays a major role in insulin regulation of metabolism in muscle, fat, and liver. Here, we show that in 3T3-L1 adipocytes, Akt operates optimally over a limited dynamic range. This indicates that Akt is a highly sensitive amplification step in the pathway. With robust insulin stimulation, substantial changes in Akt phosphorylation using either pharmacologic or genetic manipulations had relatively little effect on Akt activity. By integrating these data we observed that half-maximal Akt activity was achieved at a threshold level of Akt phosphorylation corresponding to 5-22% of its full dynamic range. This behavior was also associated with lack of concordance or demultiplexing in the behavior of downstream components. Most notably, FoxO1 phosphorylation was more sensitive to insulin and did not exhibit a change in its rate of phosphorylation between 1 and 100 nm insulin compared with other substrates (AS160, TSC2, GSK3). Similar differences were observed between various insulin-regulated pathways such as GLUT4 translocation and protein synthesis. These data indicate that Akt itself is a major amplification switch in the insulin signaling pathway and that features of the pathway enable the insulin signal to be split or demultiplexed into discrete outputs. This has important implications for the role of this pathway in disease

FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network

This study shows that Rab11 and its binding protein FIP1 are required for retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN. We also demonstrate that Golgin-97 as a FIP1-binding protein and that this binding regulates the targeting of retrograde transport vesicles to the TGN