Unbinding process of adsorbed proteins under external stress studied by atomic force microscopy spectroscopy

We report the study of the dynamics of the unbinding process under a force load f of adsorbed proteins (fibrinogen) on a solid surface (hydrophilic silica) by means of atomic force microscopy spectroscopy. By varying the loading rate rf, defined by f = rf t, t being the time, we find that, as for specific interactions, the mean rupture force increases with rf. This unbinding process is analyzed in the framework of the widely used Bell model. The typical dissociation rate at zero force entering in the model lies between 0.02 and 0.6 s−1. Each measured rupture is characterized by a force f0, which appears to be quantized in integer multiples of 180–200 pN.

Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates

Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loading rate-dependent unbinding forces were determined for single molecules by atomic force microscopy, which extrapolated at zero force to a value close to the off-rate measured in solution, without any indication for multiple transition states. The measured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indicating that the same transition state must be crossed in spontaneous and forced unbinding and that the unbinding path under load cannot be too different from the one at zero force. The distance of the transition state from the ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most likely reflects the elasticity of the protein in the unbinding process. Atomic force microscopy thus can be a valuable tool for the characterization of solution properties of protein-ligand systems at the single molecule level, predicting relative off-rates, potentially of great value for combinatorial chemistry and biology.

Chemically distinct transition states govern rapid dissociation of single L-selectin bonds under force

Carbohydrate–protein bonds interrupt the rapid flow of leukocytes in the circulation by initiation of rolling and tethering at vessel walls. The cell surface carbohydrate ligands are glycosylated proteins like the mucin P-selectin glycoprotein ligand-1 (PSGL-1), which bind ubiquitously to the family of E-, P-, and L-selectin proteins in membranes of leukocytes and endothelium. The current view is that carbohydrate–selectin bonds dissociate a few times per second, and the unbinding rate increases weakly with force. However, such studies have provided little insight into how numerous hydrogen bonds, a Ca2+ metal ion bond, and other interactions contribute to the mechanical strength of these attachments. Decorating a force probe with very dilute ligands and controlling touch to achieve rare single-bond events, we have varied the unbinding rates of carbohydrate–selectin bonds by detachment with ramps of force/time from 10 to 100,000 pN/sec. Testing PSGL-1, its outer 19 aa (19FT), and sialyl LewisX (sLeX) against L-selectin in vitro on glass microspheres and in situ on neutrophils, we found that the unbinding rates followed the same dependence on force and increased by nearly 1,000-fold as rupture forces rose from a few to ≈200 pN. Plotted on a logarithmic scale of loading rate, the rupture forces reveal two prominent energy barriers along the unbinding pathway. Strengths above 75 pN arise from rapid detachment (<0.01 sec) impeded by an inner barrier that requires a Ca2+ bond between a single sLeX and the lectin domain. Strengths below 75 pN occur under slow detachment (>0.01 sec) impeded by the outer barrier, which appears to involve an array of weak (putatively hydrogen) bonds.

Force and kinetic barriers to unzipping of the DNA double helix

A theory of the unzipping of double-stranded DNA is presented and is compared to recent micromanipulation experiments. It is shown that the interactions that stabilize the double helix and the elastic rigidity of single strands simply determine the sequence-dependent ≈12-pN force threshold for DNA strand separation. Using a semimicroscopic model of the binding between nucleotide strands, we show that the greater rigidity of the strands when formed into double-stranded DNA, relative to that of isolated strands, gives rise to a potential barrier to unzipping. The effects of this barrier are derived analytically. The force to keep the extremities of the molecule at a fixed distance, the kinetic rates for strand unpairing at fixed applied force, and the rupture force as a function of loading rate are calculated. The dependence of the kinetics and of the rupture force on molecule length is also analyzed.

Flotação aplicada ao pós-tratamento do efluente de reator anaeróbio de leito expandido tratando esgoto sanitário

Este trabalho apresenta e discute os resultados de um estudo amplo e aprofundado sobre os principais parâmetros operacionais da flotação por ar dissolvido, utilizada no pós-tratamento de efluentes de um reator anaeróbio de leito expandido (RALEx), tratando 10 m3/hora de esgoto sanitário. Foram realizados preliminarmente ensaios utilizando o flotateste, unidade de flotação em escala de laboratório, para identificar as melhores dosagens de coagulante (cloreto férrico), o polímero mais adequado, dentre os 26 testados, e sua respectiva dosagem, o pH de coagulação adequado, o tempo (Tf) e o gradiente de velocidade (Gf) de floculação mais apropriados e a quantidade de ar (S*) requerida. Para obtenção das condições operacionais adequadas para a unidade piloto de flotação, os valores de Tf e de Gf foram variados de zero a 24 minutos e de 40 a 100 s-1, respectivamente. As concentrações de cloreto férrico e de polímero sintético variaram de 15 a 92 mg/L e de 0,25 a 7,0 mg/L, respectivamente. S* variou de 2.85 a 28.5 gramas de ar por metro cúbico de efluente e a taxa de aplicação superficial na unidade de flotação abrangeu de 180 a 250 m3/m2/d. O desempenho da flotação durante a partida do reator anaeróbio também foi investigado. O uso de 50 mg/L de cloreto férrico, de Tf igual a 20 min e Gf de 80 s-1, de S* de 19,7 g de ar por m3 de efluente e taxa de 180 m3/m2/d produziu excelentes resultados nos ensaios com a instalação piloto de flotação, com elevadas remoções de carga de DQO (80,6%), de fósforo total (90,1%) e de sólidos suspensos totais (92,1%) e com turbidez entre 1,6 e 15,4 uT e residuais de ferro de 0,5 mg/L, com remoção estimada, na forma de lodo, de 77 gramas de SST por m3 de efluente tratado. Nestas mesmas condições, no sistema RALEx+FAD, foram observadas remoções globais de 91,6% de carga de DQO, de 90,1% de carga de fósforo e de 96,6% de carga de SST. O emprego da flotação por ar dissolvido (FAD) mostrou-se alternativa bastante atraente para o pós-tratamento de efluentes de reatores anaeróbios. Se a coagulação estiver bem ajustada, o sistema composto de reator anaeróbio seguido de unidade de flotação consegue alcançar excelente remoção de matéria orgânica, redução significativa da concentração de fósforo e de sólidos suspensos, além de precipitação dos sulfetos dissolvidos, gerados no reator anaeróbio. Bons resultados foram alcançados mesmo quando o reator RALEx produziu efluentes de baixa qualidade durante seu período de partida. Nesse período, o sistema de flotação atuou como barreira eficaz, evitando a emissão de efluente de baixa qualidade do sistema.

Strain and damage sensing properties on multifunctional cement composites with CNF admixture

Both strain and damage sensing properties on carbon nanofiber cement composites (CNFCC) are reported in the present paper. Strain sensing tests were first made on the material’s elastic range. The applied loading levels have been previously calculated from mechanical strength tests. The effect of several variables on the strain-sensing function was studied, e.g. cement pastes curing age, current density, loading rate or maximum stress applied. All these parameters were discussed using the gage factor as reference. After this first set of elastic experiments, the same specimens were gradually loaded until material’s failure. At the same time both strain and resistivity were measured. The former was controlled using strain gages, and the latter using a multimeter on a four probe setup. The aim of these tests was to prove the sensitivity of these CNF composites to sense their own damage, i.e. check the possibility of fabricating structural damage sensors with CNFCC’s. All samples with different CNF dosages showed good strain-sensing capacities for curing periods of 28 days. Furthermore, a 2%CNF reinforced cement paste has been sensitive to its own structural damage.

Influence of sludge retention time on filtration performance and biomass characteristics in a hollow fiber membrane bioreactor

In this study, the filtration process and the biomass characteristics in a laboratory-scale submerged membrane bioreactor (MBR) equipped with a hollow fiber (HF) microfiltration membrane were studied at different solid retention times (SRT). The MBR was fed by synthetic wastewater and the organic loading rate (OLR) was 0.5, 0.2, 0.1, and 0.08 kg COD kg VSS−1 d−1 for 10, 30, 60, and 90 days of SRT, respectively. The hydraulic retention time was 8.4 h and the permeate flux was 6 L m−2 h−1(LMH). Data analysis confirmed that at all the studied SRTs, the HF-MBR operated very good obtaining of high quality permeates. Chemical Oxygen Demand (COD) removal efficiencies were higher than 95%. The best filtration performance was reached at SRT of 30 d. On the other hand, the respirometric analysis showed that biomass was more active and there was more biomass production at low SRTs. The concentration of soluble extracellular polymeric substances (EPS) decreased with increasing SRT. A decrease of soluble EPS caused a decrease of membrane fouling rate, decreasing the frequency of chemical cleanings. The floc size decreased with SRT increasing. At high SRTs, there was more friction among particles due to the increase of the cellular density and the flocs broke decreasing their size.

Statistical tests of load-unload response ratio signals by lattice solid model: Implication to tidal triggering and earthquake prediction

Statistical tests of Load-Unload Response Ratio (LURR) signals are carried in order to verify statistical robustness of the previous studies using the Lattice Solid Model (MORA et al., 2002b). In each case 24 groups of samples with the same macroscopic parameters (tidal perturbation amplitude A, period T and tectonic loading rate k) but different particle arrangements are employed. Results of uni-axial compression experiments show that before the normalized time of catastrophic failure, the ensemble average LURR value rises significantly, in agreement with the observations of high LURR prior to the large earthquakes. In shearing tests, two parameters are found to control the correlation between earthquake occurrence and tidal stress. One is, A/(kT) controlling the phase shift between the peak seismicity rate and the peak amplitude of the perturbation stress. With an increase of this parameter, the phase shift is found to decrease. Another parameter, AT/k, controls the height of the probability density function (Pdf) of modeled seismicity. As this parameter increases, the Pdf becomes sharper and narrower, indicating a strong triggering. Statistical studies of LURR signals in shearing tests also suggest that except in strong triggering cases, where LURR cannot be calculated due to poor data in unloading cycles, the larger events are more likely to occur in higher LURR periods than the smaller ones, supporting the LURR hypothesis.

Comparison of cellulose solubilisation rates in rumen and landfill leachate inoculated reactors

The aim of this study was to conduct a number of controlled digestions to obtain easily comparable cellulose solubilisation rates and to compare these rates to those found in the literature to see which operational differences were significant in affecting cellulose degradation during anaerobic digestion. The results suggested that differences in volumetric cellulose solubilisation rates were not indicative of the true performance of cellulose digestion systems. When cellulose solubilisation rates were normalised by the mass of cellulose in the reactor at each time step, the comparison of the rates became more meaningful. Cellulose solubilisation was surface area limited. Therefore, changes in the loading rate of cellulose to the reactor altered the volumetric solubilisation rate without changing the mass normalised rate. Comparison of mass normalised solubilisation rates from this study and the literature demonstrated that differences in reactor configuration and operational conditions did not significantly impact on the solubilisation rate whereas the difference in composition of the microbial communities showed a marked effect. This work highlights the importance of using appropriately normalised data when making comparisons between systems with differing operational conditions. (c) 2005 Elsevier Ltd. All rights reserved.

Long-term flow rates and biomat zone hydrology in soil columns receiving septic tank effluent

Soil absorption systems (SAS) are used commonly to treat and disperse septic tank effluent (STE). SAS can hydraulically fail as a result of the low permeable biomat zone that develops on the infiltrative surface. The objectives of this experiment were to compare the hydraulic properties of biomats grown in soils of different textures, to investigate the long-term acceptance rates (LTAR) from prolonged application of STE, and to assess if soils were of major importance in determining LTAR. The STE was applied to repacked sand, Oxisol and Vertisol soil columns over a period of 16 months, at equivalent hydraulic loading rates of 50, 35 and 8 L/m(2)/d, respectively Infiltration rates, soil matric potentials, and biomat hydraulic properties were measured either directly from the soil columns or calculated using established soil physics theory. Biomats 1 to 2 cm thick developed in all soils columns with hydraulic resistances of 27 to 39 d. These biomats reduced a 4 order of magnitude variation in saturated hydraulic conductivity (K.) between the soils to a one order of magnitude variation in LTAR. A relationship between biomat resistance and organic loading rate was observed in all soils. Saturated hydraulic conductivity influenced the rate and extent of biomat development. However, once the biomat was established, the LTAR was governed by the resistance of the biomat and the sub-biomat soil unsaturated flow regime induced by the biomat. Results show that whilst initial soil K. is likely to be important in the establishment of the biomat zone in a trench, LTAR is determined by the biomat resistance and the unsaturated soil hydraulic conductivity, not the K, of a soil. The results call into question the commonly used approach of basing the LTAR, and ultimately trench length in SAS, on the initial K, of soils. (c) 2006 Elsevier Ltd. All rights reserved.

Mechanics of cellular adhesion to artificial artery templates

We are using polymer templates to grow artificial artery grafts in vivo for the replacement of diseased blood vessels. We have previously shown that adhesion of macrophages to the template starts the graft formation. We present a study of the mechanics of macrophage adhesion to these templates on a single cell and single bond level with optical tweezers. For whole cells, in vitro cell adhesion densities decreased significantly from polymer templates polyethylene to silicone to Tygon (167, 135, and 65 cells/mm(2)). These cell densities were correlated with the graft formation success rate (50%, 25%, and 0%). Single-bond rupture forces at a loading rate of 450 pN/s were quantified by adhesion of trapped 2-mm spheres to macrophages. Rupture force distributions were dominated by nonspecific adhesion (forces, < 40 pN). On polystyrene, preadsorption of fibronectin or presence of serum proteins in the cell medium significantly enhanced adhesion strength from a mean rupture force of 20 pN to 28 pN or 33 pN, respectively. The enhancement of adhesion by fibronectin and serum is additive (mean rupture force of 43 pN). The fraction of specific binding forces in the presence of serum was similar for polystyrene and polymethyl-methacrylate, but specific binding forces were not observed for silica. Again, we found correlation to in vivo experiments, where the density of adherent cells is higher on polystyrene than on silica templates, and can be further enhanced by fibronectin adsorption. These findings show that in vitro adhesion testing can be used for template optimization and to substitute for in-vivo experiments.

Cellulosic waste degradation by rumen-enhanced anaerobic digestion

Anaerobic digestion of lignocellulosic material is carried out effectively in many natural microbial ecosystems including the rumen. A rumen-enhanced anaerobic sequencing batch reactor was used to investigate cellulose degradation to give analysis of overall process stoichiometry and rates of hydrolysis. The reactor achieved VFA production rates of 207-236 mg COD/L/h at a loading rate of 10 g/L/d. Overloading of the reactor resulted in elevated production of propionic acid, and on occasion, the presence of succinic acid. With improvements in mixing and solids wasting, the anaerobic sequencing batch reactor system could enable full-scale application of the process for treatment of cellulosic waste material.

Crack initiation in asphalt mixtures under external compressive loads

Crack initiation was studied for asphalt mixtures under external compressive loads. High tensile localized stresses e direction of the external loads. A quantitative crack initiation criterion the edges of compressed air voids lead to the growth of wing cracks in thon was derived using pseudostrain energy balance principle. Bond energy is determined and it increases with aging and loading rate while decreases with temperature. Cohesive and adhesive cracking occur simultaneously and a method was proposed to determine the individual percentage. The crack initiation criterion is simplified and validated through comparing the predicted and measured compressive strength of the asphalt mixtures.

Clogging in subsurface-flow treatment wetlands:occurrence and contributing factors

Clogging is a major operational and maintenance issue associated with the use of subsurface flow wetlands for wastewater treatment, and can ultimately limit the lifetime of the system. This review considers over two decades of accumulated knowledge regarding clogging in both vertical and horizontal subsurface flow treatment wetlands. The various physical, chemical and biological factors responsible for clogging are identified and discussed. The occurrence of clogging is placed into the context of various design and operational parameters such as wastewater characteristics, upstream treatment processes, intermittent or continuous operation, influent distribution, and media type. This information is then used to describe how clogging develops within, and subsequently impacts, common variants of subsurface flow treatment wetland typically used in the U.S., U.K., France and Germany. Comparison of these systems emphasized that both hydraulic loading rate and solids loading rate need to be considered when designing systems to operate robustly, i.e. hydraulic overloading makes horizontal-flow tertiary treatment systems in the U.K. more susceptible to clogging problems than vertical-flow primary treatment systems in France. Future research should focus on elucidating the underlying mechanisms of clogging as they relate to the design, operation, and maintenance of subsurface flow treatment wetlands. © 2010 Elsevier B.V.

Biogeochemical Contributions of Tree Islands to Everglades Wetland Landscape Nitrogen Cycling During Seasonal Inundation

In the Florida Everglades, tree islands are conspicuous heterogeneous elements in the herbaceous wetland landscape. We characterized the biogeochemical role of a seasonally flooded tree island during wet season inundation, specifically examining hydrologically mediated flows of nitrogen (N) and N retention by the tree island. We estimated ecosystem N standing stocks and fluxes, soil and litter N transformation rates, and hydrologic fluxes of N to quantify the net ecosystem N mass flux. Results showed that hydrologic sources of N were dominated by surface water loads of nitrate (NO3) and ammonium (NH4). Nitrate immobilization by soils and surficial leaf litter was an important sink for surface water dissolved inorganic N (DIN). We estimated that the net annual DIN retention by a seasonally flooded tree island was 20.5 ± 5.0 g m−2 during wet season inundation. Based on the estimated tree island surface water DIN loading rate, a seasonally flooded tree island retained 76% of imported DIN. As such, seasonally flooded tree islands have the potential to retain 55% of DIN entering the marsh landscape via upstream canal overland flow in the wet season. By increasing reactive surface area and DOC availability, we suggest that tree islands promote convergence of elements that enhance DIN retention. Tree islands of this region are thus important components of landscape-scale restoration efforts that seek to reduce sources of anthropogenic DIN to downstream estuaries.