High aeration rate enhances flow stratification in full-scale oxidation ditch

Aerated channel reactors with a uniform field of aeration may display flow stratification and short-circuit phenomena in wastewater treatment systems. In this study, we present data suggesting that flow stratification is closely related to the aeration rate and the arrangement of aerators. A full-scale oxidation ditch, with a total volume of 6,500 m³ and a membrane-diffused aerated zone of 60 × 7 × 5 m (length–width–depth), was selected for water velocity measurements. Two profiles of the oxidation ditch were studied in detail: the first one was at the end of the aerated zone and the second one at the end of the anoxic zone. The results of this work demonstrate that the horizontal water velocity at the end of the aerated zone displayed significant stratification, with maximum velocity near the water surface (0.5–0.7 m/s) and almost zero velocity at a depth of 2.5 m. At the end of the anoxic zone, water velocity was uniform and equal to 0.27–0.31 m/s. Increasing the aeration rate from 1,800 to 4,300 m³/h, almost 90% of the water flow was found to discharge through the upper-half of the channel reactor profile. Different options to mitigate flow stratification of the oxidation ditch are discussed in this paper.     

Single-Molecule Force Spectroscopy Study on the Mechanism of RNA Disassembly in Tobacco Mosaic Virus

To explore the disassembly mechanism of tobacco mosaic virus (TMV), a model system for virus study, during infection, we have used single-molecule force spectroscopy to mimic and follow the process of RNA disassembly from the protein coat of TMV by the replisome (molecular motor) in vivo, under different pH and Ca²⁺ concentrations. Dynamic force spectroscopy revealed the unbinding free-energy landscapes as that at pH 4.7 the disassembly process is dominated by one free-energy barrier, whereas at pH 7.0 the process is dominated by one barrier and that there exists a second barrier. The additional free-energy barrier at longer distance has been attributed to the hindrance of disordered loops within the inner channel of TMV, and the biological function of those protein loops was discussed. The combination of pH increase and Ca²⁺ concentration drop could weaken RNA-protein interactions so much that the molecular motor replisome would be able to pull and disassemble the rest of the genetic RNA from the protein coat in vivo. All these facts provide supporting evidence at the single-molecule level, to our knowledge for the first time, for the cotranslational disassembly mechanism during TMV infection under physiological conditions.     

Theoretical Investigation of Interaction of Sorbitol Molecules with Alcohol Dehydrogenase in Aqueous Solution Using Molecular Dynamics Simulation

The nature of protein–sorbitol–water interaction in solution at the molecular level, has been investigated using molecular dynamics simulations. In order to do this task, two molecular dynamics simulations of the protein ADH in solution at room temperature have been carried out, one in the presence (about 0.9 M) and another in the absence of sorbitol. The results show that the sorbitol molecules cluster and move toward the protein, and form hydrogen bonds with protein. Also, coating by sorbitol reduces the conformational fluctuations of the protein compared to the sorbitol-free system. Thus, it is concluded that at moderate concentration of sorbitol solution, sorbitol molecules interact with ADH via many H-bonds that prevent the protein folding. In fact, at more concentrated sorbitol solution, water and sorbitol molecules accumulate around the protein surface and form a continuous space-filling network to reduce the protein flexibility. Namely, in such solution, sorbitol molecules can stabilize a misfolded state of ADH, and prevent the protein from folding to its native structure.     

Long-range DNA-water interactions

DNA functions only in aqueous environments and adopts different conformations depending on the hydration level. The dynamics of hydration water and hydrated DNA leads to rotating and oscillating dipoles that, in turn, give rise to a strong megahertz to terahertz absorption. Investigating the impact of hydration on DNA dynamics and the spectral features of water molecules influenced by DNA, however, is extremely challenging because of the strong absorption of water in the megahertz to terahertz frequency range. In response, we have employed a high-precision megahertz to terahertz dielectric spectrometer, assisted by molecular dynamics simulations, to investigate the dynamics of water molecules within the hydration shells of DNA as well as the collective vibrational motions of hydrated DNA, which are vital to DNA conformation and functionality. Our results reveal that the dynamics of water molecules in a DNA solution is heterogeneous, exhibiting a hierarchy of four distinct relaxation times ranging from ∼8 ps to 1 ns, and the hydration structure of a DNA chain can extend to as far as ∼18 Å from its surface. The low-frequency collective vibrational modes of hydrated DNA have been identified and found to be sensitive to environmental conditions including temperature and hydration level. The results reveal critical information on hydrated DNA dynamics and DNA-water interfaces, which impact the biochemical functions and reactivity of DNA.     

Dioxygen and nitric oxide pathways and affinity to the catalytic site of rubredoxin:oxygen oxidoreductase from Desulfovibrio gigas

Rubredoxin:oxygen oxidoreductase (ROO) is the terminal oxidase of a soluble electron transfer chain found in Desulfovibrio gigas. This protein belongs to the flavodiiron family and was initially described as an oxygen reductase, converting this substrate to water and acting as an oxygen-detoxifying system. However, more recent studies evidenced also the ability for this protein to act as a nitric oxide reductase, suggesting an alternative physiological role. To clarify the apparent bifunctional nature of this protein, we performed molecular dynamics simulations of the protein, in different redox states, together with O₂ and NO molecules in aqueous solution. The two small molecules were parameterized using free-energy calculations of the hydration process. With these simulations we were able to identify specific protein paths that allow the diffusion of both these molecules through the protein towards the catalytic centers. Also, we have tried to characterize the preference of ROO towards the presence of O₂ and/or NO at the active site. By using free-energy simulations, we did not find any significant preference for ROO to accommodate both O₂ and NO. Also, from our molecular dynamics simulations we were able to identify similar diffusion profiles for both O₂ and NO molecules. These two conclusions are in good agreement with previous experimental works stating that ROO is able to catalyze both O₂ and NO. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Metabolic relationship between invertase and acid phosphatase isoenzymes in Saccharomyces cerevisiae

Repressed cells of Saccharomyces cerevisiae, subjected to inhibition of both RNA and protein synthesis, showed a pattern of membrane-bound and cytosol acid phosphatase to the external enzyme which seemed to be linked through a precursor-product relationship.Gel exclusion chromatography did not indicate clear differences between the isoenzymes. Moreover, centrifugation experiments in CsCl and precipitation with concanavalin A suggested that there were no acid phosphatase molecules devoid of carbohydrate. Membrane-bound invertase displayed a molecular weight and a carbohydrate to protein ratio smaller than those of the exocellular enzyme. The values of molecular weight and buoyant density of the membrane-bound enzyme were closer to those found for the cytosol invertase. The stability of the level of the soluble invertase detected in the cytoplasm under derepression conditions, or after RNA or protein synthesis inhibition was found to be only apparent and represented the result of an equilibrium between synthesis and degradation.     

Molecular-level thermodynamic and kinetic parameters for the self-assembly of apoferritin molecules into crystals

The self-assembly of apoferritin molecules into crystals is a suitable model for protein crystallization and aggregation; these processes underlie several biological and biomedical phenomena, as well as for protein and virus self-assembly. We use the atomic force microscope in situ, during the crystallization of apoferritin to visualize and quantify at the molecular level the processes responsible for crystal growth. To evaluate the governing thermodynamic parameters, we image the configuration of the incorporation sites, "kinks", on the surface of a growing crystal. We show that the kinks are due to thermal fluctuations of the molecules at the crystal-solution interface. This allows evaluation of the free energy of the intermolecular bond phi=3.0 k(B)T=7.3 kJ/mol. The crystallization free energy, extracted from the protein solubility, is -42 kJ/mol. Published determinations of the second virial coefficient and the protein solubility between 0 and 40 degrees C revealed that the enthalpy of crystallization is close to zero. Analyses based on these three values suggest that the main component in the crystallization driving force is the entropy gain of the water molecules bound to the protein molecules in solution and released upon crystallization. Furthermore, monitoring the incorporation of individual molecules in to the kinks, we determine the characteristic frequency of attachment of individual molecules at one set of conditions. This allows a correlation between the mesoscopic kinetic coefficient for growth and the molecular-level thermodynamic and kinetic parameters determined here. We found that step growth velocity, scaled by the molecular size, equals the product of the kink density and attachment frequency, i.e. the latter pair are the molecular-level parameters for self-assembly of the molecules into crystals.     

Enhanced crystal packing due to solvent reorganization through reductive methylation of lysine residues in oxidoreductase from <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>

Protein crystallization is in part driven by the changes in the entropy of the system, but opinions differ as to whether the solute (protein) or solvent (water) molecules make more of a contribution to the overall entropic change. Methylation of lysine residues in proteins has been used to enhance protein crystallization. We investigated using molecular dynamics simulations with explicit solvent molecules, the behavior of several native proteins and their methylated counterparts chosen from an earlier large-scale study. Methylated lysines are capable of making a variety of interactions including H-bonds with protein residues and solvent. We demonstrate that methylation on the lysine slightly increases its side chain conformational entropy by about 3.5 J mol⁻¹ K⁻¹. Analysis of the radial and spatial distributions of the water molecules around the methylated lysine surface in oxidoreductase from Streptococcus pneumoniae revealed a larger sphere of water molecules with low entropy, as compared with solvent associated with unmethylated lysine. If methylated lysine were to make interactions at the protein–protein interface, the low-entropy water molecules associated with methylated lysines would be released, resulting in a gain of entropy. We show that this gain more than compensates for the loss of protein entropy. Therefore, we propose that lysine methylation favors the formation of crystals through solvent entropic gain.     

Effect of thermo-velocity barriers on fish: influence of water temperature,flow velocity and body size on the volitional swimming capacity of northern straight-mouth nase (Pseudochondrostoma duriense)

Water temperature and flow velocity directly affect the fish swimming capacity, and thus, both variables influence the fish passage through river barriers. Nonetheless, their effects are usually disregarded in fishway engineering and management. This study aims to evaluate the volitional swimming capacity of the northern straight-mouth nase (Pseudochondrostoma duriense), considering the possible effects of water temperature, flow velocity and body size. For this, the maximum distance, swim speed and fatigue time (FT) were studied in an outdoor open-channel flume in the Duero River (Burgos, Spain) against three nominal velocities (1.5, 2.5 and 3 m s⁻¹) and temperatures (5.5, 13.5 and 18.5°C), also including the changes between swimming modes (prolonged and sprint). Results showed that a nase of 20.8 cm mean fork length can develop a median swim speed that exceeds 20.7 BL s⁻¹ (4.31 m s⁻¹) during a median time of 3.4 s in sprint mode, or 12.2 BL s⁻¹ (2.55 m s⁻¹) for 23.7 s in prolonged mode under the warmest scenario. During prolonged swimming mode, fish were able to reach further distances in warmer water conditions for all situations, due to a greater swimming speed and FT, whereas during sprint mode, warmer conditions increased the swim speed maintaining the FT. In conclusion, the studied temperature range and flow velocity range influence fish swimming performance, endurance and distance travelled, although with some differences depending on the swimming mode. The provided information goes a step forward in the definition of real fish swimming capacities, and in turn, will contribute to establish clear passage criteria for thermo-velocity barriers, allowing the calculation of the proportion of fish able to pass a barrier under different working scenarios, as well designing of the optimized solutions to improve the fish passage through river barriers.     

Locating missing water molecules in protein cavities by the three-dimensional reference interaction site model theory of molecular solvation

Water molecules confined in protein cavities are of great importance in understanding the protein structure and functions. However, it is a nontrivial task to locate such water molecules in protein by the ordinary molecular simulation and modeling techniques as well as experimental methods. The present study proves that the three-dimensional reference interaction site model (3D-RISM) theory, a recently developed statistical-mechanical theory of molecular solvation, has an outstanding advantage in locating such water molecules. In this paper, we demonstrate that the 3D-RISM theory is able to reproduce the structure and the number of water molecules in cavities of hen egg-white lysozyme observed commonly in the X-ray structures of different resolutions and conditions. Furthermore, we show that the theory successfully identified a water molecule in a cavity, the existence of which has been ambiguous even from the X-ray results. In contrast, we confirmed that molecular dynamics simulation is helpless at present to find such water molecules because the results substantially depend on the initial coordinates of water molecules. Possible applications of the theory to problems in the fields of biochemistry and biophysics are also discussed.     

Single-molecule force spectroscopy of cartilage aggrecan self-adhesion

We investigated self-adhesion between highly negatively charged aggrecan macromolecules extracted from bovine cartilage extracellular matrix by performing atomic force microscopy (AFM) imaging and single-molecule force spectroscopy (SMFS) in saline solutions. By controlling the density of aggrecan molecules on both the gold substrate and the gold-coated tip surface at submonolayer densities, we were able to detect and quantify the Ca²⁺-dependent homodimeric interaction between individual aggrecan molecules at the single-molecule level. We found a typical nonlinear sawtooth profile in the AFM force-versus-distance curves with a molecular persistence length of l_p = 0.31 ± 0.04 nm. This is attributed to the stepwise dissociation of individual glycosaminoglycan (GAG) side chains in aggrecans, which is very similar to the known force fingerprints of other cell adhesion proteoglycan systems. After studying the GAG-GAG dissociation in a dynamic, loading-rate-dependent manner (dynamic SMFS) and analyzing the data according to the stochastic Bell-Evans model for a thermally activated decay of a metastable state under an external force, we estimated for the single glycan interaction a mean lifetime of τ = 7.9 ± 4.9 s and a reaction bond length of x_β = 0.31 ± 0.08 nm. Whereas the x_β-value compares well with values from other cell adhesion carbohydrate recognition motifs in evolutionary distant marine sponge proteoglycans, the rather short GAG interaction lifetime reflects high intermolecular dynamics within aggrecan complexes, which may be relevant for the viscoelastic properties of cartilage tissue.     

Habitat preferences of the burbot (Lota lota) from the River Elbe: an experimental approach

An experimental approach was used to analyze preferences of the burbot (Lota lota) with regard to water depth, substrate type and flow velocity. In total, 30 burbots used in the experiments were caught in the middle reaches of the River Elbe by electro‐fishing. Immediately after capture, they were placed in a 180 L transportation box with aerated freshwater and taken to the research aquarium of the Zoological Museum Hamburg where they were divided into two groups according to total length: group I, 10.0–16.5 cm; group II, 20.0–30.0 cm. Prior to the experiments the fish were adapted to laboratory conditions for four weeks at 20°C in four tanks, each containing 720 L water. For each experiment, three L. lota were transferred to an additional 1350 L experimental tank. After a one‐day adaptation phase each experiment ran for three continuous day and night phases. Burbot preferences were tested for (i) water depth, (ii) substrate size, (iii) water depth and substrate size combined, and (iv) water depth, substrate and flow velocity in combination. Diurnal and nocturnal activities were recorded and analyzed throughout all experiments. Burbots of both length groups showed a significant preference for deeper water depths and large cobble substrate. Substrate type was found to be the dominant factor determining habitat preferences of L. lota when investigated in combination with water depth. In experiments combined with flow velocity, substrate and water depth were the most important factors determining burbot habitat preferences, and flow velocity of minor importance. In general, burbots were most active at night, while daytime activity was much lower. However, in all experimental approaches in the smaller length group the burbots displayed much higher day‐ and night‐time activity than did the individuals in the larger length group.     

Vegetation as self‐adaptive coastal protection: Reduction of current velocity and morphologic plasticity of a brackish marsh pioneer

By reducing current velocity, tidal marsh vegetation can diminish storm surges and storm waves. Conversely, currents often exert high mechanical stresses onto the plants and hence affect vegetation structure and plant characteristics. In our study, we aim at analysing this interaction from both angles. On the one hand, we quantify the reduction of current velocity by Bolboschoenus maritimus, and on the other hand, we identify functional traits of B. maritimus’ ramets along environmental gradients. Our results show that tidal marsh vegetation is able to buffer a large proportion of the flow velocity at currents under normal conditions. Cross‐shore current velocity decreased with distance from the marsh edge and was reduced by more than 50% after 15 m of vegetation. We were furthermore able to show that plants growing at the marsh edge had a significantly larger diameter than plants from inside the vegetation. We found a positive correlation between plant thickness and cross‐shore current which could provide an adaptive value in habitats with high mechanical stress. With the adapted morphology of plants growing at the highly exposed marsh edge, the entire vegetation belt is able to better resist the mechanical stress of high current velocities. This self‐adaptive effect thus increases the ability of B. maritimus to grow and persist in the pioneer zone and may hence better contribute to ecosystem‐based coastal protection by reducing current velocity.     

Characteristics of surface-water flows in the ridge and slough landscape of Everglades National Park: implications for particulate transport

Over the last one hundred years, compartmentalization and water management activities have reduced water flow to the ridge and slough landscape of the Everglades. As a result, the once corrugated landscape has become topographically and vegetationally uniform. The focus of this study was to quantify variation in surface flow in the ridge and slough landscape and to relate flow conditions to particulate transport and deposition. Over the 2002–2003 and 2003–2004 wet seasons, surface velocities and particulate accumulation were measured in upper Shark River Slough in Everglades National Park. Landscape characteristics such as elevation, plant density and biomass also were examined to determine their impact on flow characteristics and material transport. The results of this study demonstrate that the release of water during the wet season not only increases water levels, but also increased flow speeds and particulate transport and availability. Further, flow speeds were positively and significantly correlated with water level thereby enhancing particulate transport in sloughs relative to ridges especially during peak flow periods. Our results also indicate that the distribution of biomass in the water column, including floating plants and periphyton, affects velocity magnitude and shape of vertical profiles, especially in the sloughs where Utricularia spp. and periphyton mats are more abundant. Plot clearing experiments suggest that the presence of surface periphyton and Utricularia exert greater control over flow characteristics than the identity (i.e., sawgrass or spike rush) or density of emergent macrophytes, two parameters frequently incorporated into models describing flow through vegetated canopies. Based on these results, we suggest that future modeling efforts must take the presence of floating biomass, such as Utricularia, and presence of periphyton into consideration when describing particulate transport.     

Mean flow and turbulence characteristics of a full-scale spiral corrugated culvert with implications for fish passage

A micro-acoustic Doppler velocimeter (ADV) was used to measure three-dimensional mean velocity and turbulence characteristics in a full-scale culvert with spiral corrugations. The culvert was set up in a test bed constructed to examine juvenile salmon passage success in various culvert types. The test culvert was 12.2 m long and 1.83 m in diameter and set at a 1.14% slope. The corrugations were 2.54 cm deep by 7.62 cm peak to peak with a 5° right-handed pitch. Cross-sectional grids of ADV measurements were taken at discharges of 0.028, 0.043, 0.071, 0.099, 0.113, 0.227, and 0.453 m³/s at nine locations. In the uniform flow region, the centerline velocity profiles were consistent with fully rough turbulent flows and the friction factor was independent of Reynolds number and was very close to theoretical results. Secondary flow induced by the spiral corrugations caused asymmetries in the velocity and turbulence distributions creating a reduced velocity zone (RVZ) on the right side of the culvert as seen looking upstream, which small fish could utilize to aid their upstream passage. Velocity and axial components of turbulence in the RVZ were found to be much less than in mid-channel or on the left of the culvert, and the difference became greater at increased flow rates. In addition, cross-stream and vertical velocity components within the RVZ were small relative to the downstream axial component, while lateral and vertical turbulence intensities were comparable to the axial component. Observations from a concurrent fish passage study showed that more juvenile fish migrate through the right side of the culvert within the RVZ.     

Modeling structure and flexibility of <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B in organic solvents

Background  

The structure and flexibility of Candida antarctica lipase B in water and five different organic solvent models was investigated using multiple molecular dynamics simulations to describe the effect of solvents on structure and dynamics. Interactions of the solvents with the protein and the distribution of water molecules at the protein surface were examined.     

Active-site hydration and water diffusion in cytochrome P450cam: a highly dynamic process

Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.     

Pressure-Induced Changes in the Structure and Function of the Kinesin-Microtubule Complex

Kinesin-1 is an ATP-driven molecular motor that “walks” along a microtubule by working two heads in a “hand-over-hand” fashion. The stepping motion is well-coordinated by intermolecular interactions between the kinesin head and microtubule, and is sensitively changed by applied forces. We demonstrate that hydrostatic pressure works as an inhibitory action on kinesin motility. We developed a high-pressure microscope that enables the application of hydrostatic pressures of up to 200 MPa (2000 bar). Under high-pressure conditions, taxol-stabilized microtubules were shortened from both ends at the same speed. The sliding velocity of kinesin motors was reversibly changed by pressure, and reached half-maximal value at ∼100 MPa. The pressure-velocity relationship was very close to the force-velocity relationship of single kinesin molecules, suggesting a similar inhibitory mechanism on kinesin motility. Further analysis showed that the pressure mainly affects the stepping motion, but not the ATP binding reaction. The application of pressure is thought to enhance the structural fluctuation and/or association of water molecules with the exposed regions of the kinesin head and microtubule. These pressure-induced effects could prevent kinesin motors from completing the stepping motion.     

Flow velocity affects internal oxygen conditions in the seagrass Cymodocea nodosa

The internal oxygen status of seagrass tissues, which is believed to play an important role in events of seagrass die-off, is partly determined by the rates of gas exchange between leaves and water column. In this study, we examined whether water column flow velocity has an effect on gas exchange, and hence on internal oxygen partial pressures (pO₂) in the Mediterranean seagrass, Cymodocea nodosa. We measured the internal pO₂ in the horizontal rhizomes of C. nodosa in darkness at different mainstream flow velocities, combined with different levels of water column oxygen pO₂ using an experimental flume in the laboratory. Flow velocity clearly had an effect on the internal oxygen status. In stagnant, but fully aerated water the mean internal pO₂ was 6.9 kPa, corresponding to about 30% of air saturation. The internal pO₂ increased with increasing flow velocity reaching saturation of around 12.2 kPa (60% of air saturation) at flow velocities ≥7 cm s⁻¹. Flow had a relatively larger influence on internal pO₂ at lower water column oxygen concentrations. By extrapolating linear relationships between internal and water column pO₂ in this experimental setup, rhizomes would become anoxic at a water column oxygen pO₂ of 4–4.5 kPa (∼20% of air saturation) in flowing water, but already at 6.4 kPa (∼30% of air saturation) in stagnant water. Water flow may play an important role for seagrass performance and survival in areas with poor water column oxygen conditions and may, in general, be of importance for the distribution of submerged rooted plants.     

Kinesin velocity increases with the number of motors pulling against viscoelastic drag

Although the properties of single kinesin molecular motors are well understood, it is not clear whether multiple motors pulling a single vesicle in a cell cooperate or interfere with one another. To learn how small numbers of motors interact, microtubule gliding assays were carried out with full-length Drosophila kinesin in a novel motility medium containing xanthan, a stiff, water-soluble polysaccharide. At 2 mg/ml xanthan, the zero-shear viscosity of this medium is 1,000 times the viscosity of water, similar to cellular viscosity. To mimic the rheological drag force on the motors when attached to a vesicle in a cell, we attached a 2 μm bead to one end of the microtubule (MT). During gliding assays in our novel medium, the moving bead exerted a drag force of 4–15 pN on the kinesins pulling the MT. The velocity of MTs with an attached bead increased with MT length and with kinesin concentration. The increase with MT length arose because the number of motors is directly proportional to MT length. Our results show that small numbers of kinesins cooperate constructively when pulling against a viscoelastic drag. In the absence of a bead but still in the viscous medium, MT velocity was independent of MT length and kinesin concentration because the thin MT, like a snake moving through grass, was able to move between xanthan molecules with little resistance. A minimal shared-load model in which the number of motors is proportional to MT length fits the observed dependence of gliding velocity on MT length and kinesin concentration.