Evaluation of Persistent Lymphatic Fluid Leakage Using a Strategy of Placing a Drain After Kidney Transplantation: A Statistical Analysis to Assess Its Origin.

OBJECTIVES: Using a strategy of placing a surgical drain after kidney transplantation, the duration of a lymphatic fluid leakage and prevalence of a symptomatic lymphocele were retrospectively analyzed. The risk factors for persistent lymphatic fluid leakage or asymptomatic lymphocele were evaluated using multivariate analysis to estimate the origin of the lymphatic fluid leakage. MATERIALS AND METHODS: Patients with persistent lymphatic fluid leakage and symptomatic lymphocele were defined as those with lymphatic fluid drainage >50 mL for more than 15 days and those who required a percutaneous drainage of the lymphocele, respectively. RESULTS: Persistent lymphatic fluid leakage and symptomatic lymphocele were observed in 40 (16.4%) and 10 (4.1%) of a total of 244 patients, respectively. The maximum durations of lymphatic fluid drainage from the initial drain tube and the second drainage of the symptomatic lymphocele were 48 and 28 days, respectively. Anastomosis of the graft artery to the external iliac artery was an independent risk factor to predict persistent lymphatic fluid leakage or symptomatic lymphocele after kidney transplantation (odds = 2.597, P = .008). CONCLUSION: The findings of the study suggest that the lymphatic fluid originates from the recipient's iliac lymph trunk rather than from the graft kidney.

Diet-induced alteration of fatty acid synthase in prostate cancer progression.

Fatty acid synthase (FASN) is a cytosolic metabolic enzyme that catalyzes de novo fatty acid synthesis. A high-fat diet (HFD) is attributed to prostate cancer (PCa) progression, but the role FASN on HFD-mediated PCa progression remains unclear. We investigated the role of FASN on PCa progression in LNCaP xenograft mice fed with HFD or low-fat diet (LFD), in PCa cells, and in clinical PCa. The HFD promoted tumour growth and FASN expression in the LNCaP xenograft mice. HFD resulted in AKT and extracellular signal-regulated kinase (ERK) activation and 5' adenosine monophosphate-activated protein kinase (AMPK) inactivation. Serum FASN levels were significantly lower in the HFD group (P=0.026) and correlated inversely with tumour volume (P=0.022). Extracellular FASN release was enhanced in the PCa cells with phosphatidylinositol 3-kinase (PI3K)/mitogen-activated protein kinase (MAPK) inhibition and AMPK signalling activation. FASN inhibition resulted in decrease of PCa cell proliferation through PI3K/MAPK downregulation and AMPK activation. Furthermore, AMPK activation was associated with FASN downregulation and PI3K/MAPK inactivation. Clinically, high FASN expression was significantly associated with high Gleason scores and advanced pathological T stage. Moreover, FASN expression was markedly decreased in the PCa response to androgen deprivation therapy and chemotherapy. HFD modulates FASN expression, which may be an important mechanism in HFD-associated PCa progression. Furthermore, a critical stimulatory loop exists between FASN and the PI3K/MAPK system, whereas AMPK signalling was associated with suppression. These may offer appropriate targets for chemoprevention and cancer therapy in HFD-induced PCa.

High-level semi-synthetic production of the potent antimalarial artemisinin.

In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths. The World Health Organization has recommended artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by the parasite Plasmodium falciparum. Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties, produced by the plant Artemisia annua. However, the supply of plant-derived artemisinin is unstable, resulting in shortages and price fluctuations, complicating production planning by ACT manufacturers. A stable source of affordable artemisinin is required. Here we use synthetic biology to develop strains of Saccharomyces cerevisiae (baker's yeast) for high-yielding biological production of artemisinic acid, a precursor of artemisinin. Previous attempts to produce commercially relevant concentrations of artemisinic acid were unsuccessful, allowing production of only 1.6 grams per litre of artemisinic acid. Here we demonstrate the complete biosynthetic pathway, including the discovery of a plant dehydrogenase and a second cytochrome that provide an efficient biosynthetic route to artemisinic acid, with fermentation titres of 25 grams per litre of artemisinic acid. Furthermore, we have developed a practical, efficient and scalable chemical process for the conversion of artemisinic acid to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for specialized photochemical equipment. The strains and processes described here form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization into active pharmaceutical ingredients (for example, artesunate) for incorporation into ACTs. Because all intellectual property rights have been provided free of charge, this technology has the potential to increase provision of first-line antimalarial treatments to the developing world at a reduced average annual price.

Limited sampling strategies to estimate the area under the concentration-time curve. Biases and a proposed more accurate method.

BACKGROUND: Over 100 limited sampling strategies (LSSs) have been proposed to reduce the number of blood samples necessary to estimate the area under the concentration-time curve (AUC). The conditions under which these strategies succeed or fail remain to be clarified. OBJECTIVES: We investigated the accuracy of existing LSSs both theoretically and numerically by Monte Carlo simulation. We also proposed two new methods for more accurate AUC estimations. METHODS: We evaluated the following existing methods theoretically: i) nonlinear curve fitting algorithm (NLF), ii) the trapezium rule with exponential curve approximation (TZE), and iii) multiple linear regression (MLR). Taking busulfan (BU) as a test drug, we generated a set of theoretical concentration-time curves based on the identified distribution of pharmacokinetic parameters of BU and re-evaluated the existing LSSs using these virtual validation profiles. Based on the evaluation results, we improved the TZE so that unrealistic parameter values were not used. We also proposed a new estimation method in which the most likely curve was selected from a set of pre-generated theoretical concentration-time curves. RESULTS: Our evaluation, based on clinical profiles and a virtual validation set, revealed: i) NLF sometimes overestimated the absorption rate constant Ka, ii) TZE overestimated AUC over 280% when Ka is small, and iii) MLR underestimated AUC over 30% when the elimination rate constant Ke is small. These results were consistent with our mathematical evaluations for these methods. In contrast, our two new methods had little bias and good precision. CONCLUSIONS: Our investigation revealed that existing LSSs induce different but specific biases in the estimation of AUC. Our two new LSSs, a modified TZE and one using model concentration-time curves, provided accurate and precise estimations of AUC.

Experimental approaches for solution X-ray scattering and fiber diffraction.

X-ray scattering and diffraction from non-crystalline systems have gained renewed interest in recent years, as focus shifts from the structural chemistry information gained by high-resolution studies to the context of structural physiology at larger length scales. Such techniques permit the study of isolated macromolecules as well as highly organized macromolecular assemblies as a whole under near-physiological conditions. Time-resolved approaches, made possible by advanced synchrotron instrumentation, add a crucial dimension to many of these investigations. This article reviews experimental approaches in non-crystalline X-ray scattering and diffraction that may be used to illuminate important scientific questions such as protein/nucleic acid folding and structure-function relationships in large macromolecular assemblies.

Regional inventory of soil surface nitrogen balances in Indian agriculture (2000-2001).

Nitrogen regulates several ecological and biogeochemical processes and excess reactive nitrogen in the environment can lead to pollution problems, including the deterioration of air quality, disruption of forest processes, acidification of lakes and streams, and degradation of coastal waters. Much of the excess nitrogen inputs are related to food and energy production. An important step to understanding the sources of nitrogen and ultimately defining solutions to excess nitrogen is to describe the geographic distribution of agricultural nitrogen contributions from different regions. In this study, soil surface nitrogen loads were quantified for different states of India for the period 2000-2001. Nearly 35.4 Tg of nitrogen has been estimated as inputs from different sources, with output nitrogen from harvested crops of about 21.20 Tg. The soil surface nitrogen balance, estimated as inputs minus outputs, is found to be about 14.4 Tg surplus from the agricultural land of India. Livestock manure constituted a major percentage of total inputs (44.06%), followed by inorganic fertilizer (32.48%), atmospheric deposition (11.86%) and nitrogen fixation (11.58%). Nitrogen balance varied from deficit to surplus for different states. The highest nitrogen surplus was found in Uttar Pradesh (2.50 Tg) followed by Madhya Pradesh (1.83 Tg), Andhra Pradesh (1.79 Tg), etc. A negative nitrogen balance was found in Orissa (-0.01 Tg), Andaman Nicobar Islands (-0.32 Tg) and for some of the northeastern states. Major fertilizer consumption states were found to be Tamilnadu (204 kg/ha), Haryana (132 kg/ha), Punjab (148 kg/ha), followed by others. Similarly, nitrogen inputs from total livestock excretions were found to be high for Kerala (616 kg/ha), Jammu and Kashmir (389 kg/ha), Tamil Nadu (338 kg/ha), etc. The average nitrogen surplus of about 54 kg/ha observed for the agricultural land of the entire country of India is comparatively higher than the average surplus of about 31 kg/ha reported for European countries. These results, obtained from nutrient mass balance calculations, will be useful to formulate nutrient management plans relating to fertilizer usage, livestock management and for adopting some best management strategies at a state level in India.

Nutrient regulation of epothilone biosynthesis in heterologous and native production strains.

Fermentation media with different initial concentrations of ammonium and phosphate salts were used to study the inhibitory effects of those ions on growth and production of epothilone in Sorangium cellulosum and Myxococcus xanthus. The native epothilone producer, S. cellulosum was more sensitive to ammonium and phosphate than the heterologous producer, M. xanthus. An ammonium concentration of 12 mM reduced epothilone titers by 90% in S. cellulosum but by only 40% in M. xanthus. When 5 mM phosphate was added to the medium, production in both strains was 60% lower. Higher phosphate concentrations had little additional effect on M. xanthus titers, but epothilone production with 17 mM extra-cellular phosphate in S. cellulosum was 95% lower than in the control condition. The effect of iron supplementation to the fermentation medium was also investigated. Both strains showed best production with 20 microM iron added to the medium.

Seasonal changes of CO(2), CH(4) and N(2)O fluxes in relation to land-use change in tropical peatlands located in coastal area of South Kalimantan.

Tropical peatland could be a source of greenhouse gases emission because it contains large amounts of soil carbon and nitrogen. However these emissions are strongly influenced by soil moisture conditions. Tropical climate is characterized typically by wet and dry seasons. Seasonal changes in the emission of carbon dioxide (CO(2)), methane (CH(4)) and nitrous oxide (N(2)O) were investigated over a year at three sites (secondary forest, paddy field and upland field) in the tropical peatland in South Kalimantan, Indonesia. The amount of these gases emitted from the fields varied widely according to the seasonal pattern of precipitation, especially methane emission rates were positively correlated with precipitation. Converting from secondary forest peatland to paddy field tended to increase annual emissions of CO(2) and CH(4) to the atmosphere (from 1.2 to 1.5 kg CO(2)-C m(-2)y(-1) and from 1.2 to 1.9 g CH(4)-C m(-2)y(-1)), while changing land-use from secondary forest to upland tended to decrease these gases emissions (from 1.2 to 1.0 kg CO(2)-C m(-2)y(-1) and from 1.2 to 0.6 g CH(4)-C m(-2)y(-1)), but no clear trend was observed for N(2)O which kept negative value as annual rates at three sites.

Surface flux measurements of CO2 and N2O from a dried rice paddy in Japan during a fallow winter season.

The CO2 and N2O soil emissions at a rice paddy in Mase, Japan, were measured by enclosures during a fallow winter season. The Mase site, one of the AsiaFlux Network sites in Japan, has been monitored for moisture, heat, and CO2 fluxes since August 1999. The paddy soil was found to be a source of both CO2 and N2O flux from this experiment. The CO2 and N2O fluxes ranged from -27.6 to 160.4 microg CO2/m2/sec (average of 49.1 +/- 42.7 microg CO2/m2/sec) and from -4.4 to 129.5 ng N2O/m2/sec (average of 40.3 +/- 35.6 ng N2O/m2/ sec), respectively. A bimodal trend, which has a sub-peak in the morning around 10:00 a.m. and a primary peak between 2:00 and 3:00 p.m., was observed. Gas fluxes increased with soil temperature, but this temperature dependency seemed to occur only on the calm days. Average CO2 and N2O fluxes were 27.7 microg CO2/m2/sec and 13.4 ng N2O/m2/sec, with relatively small fluctuation during windy days, while averages of 69.3 microg CO2/m2/sec and 65.8 ng N2O/m2/sec were measured during calm days. This relationship was thought to be a result of strong surface winds, which enhance gas exchange between the soil surface and the atmosphere, thus reducing the gas emissions from soil surfaces.

Simulation of potassium extraction by continuous haemodiafiltration.

Prediction of potassium extraction rates by continuous haemodiafiltration (CHDF) is useful for safe management of potassium levels in patients with hyperkalaemia. For this purpose, we developed a formula to predict the quantity of potassium extracted by CHDF. We hypothesized that potassium concentration in efflux dialysate was completely saturated by the influx blood, and potassium concentration in efflux blood was calculated based on this hypothesis. To check the accuracy of the calculation, potassium concentration was measured in efflux blood, and values were compared to predicted concentrations. Predicted potassium concentrations demonstrated good correlation to the measured values (95% confidence range, -0.32 to 0.58 mmol/l). These results confirmed that our hypothesis is applicable to clinical use of CHDF. Based on our observations, we created a formula to accurately predict the amount of potassium (dK) extracted by CHDF: dK=(Cb-Cd)xQd+CbxQf-CsxQs (Cb: plasma potassium concentration; Cd: potassium concentration in a dialysate; Cs: potassium concentration in the replacement fluid; Qd: dialysis rate; Qf: filtration rate and Qs: infusion rate of replacement fluid).

Modulation of epothilone analog production through media design.

Recently, the epothilone polyketide synthase (PKS) was successfully introduced into a heterologous production host for the large-scale production of epothilone D. We have found that at least three other epothilones can also be produced as the major fermentation product of this recombinant strain by supplementation of specific substrates to the production media. Addition of acetate or propionate to the media results in modulation of the epothilone D:C ratio, whereas addition of L-serine with either acetate or propionate yields epothilone H(1) or H(2) as the major product. This strategy permits production of at least four novel epothilones by culturing a single host with a genetically modified epothilone PKS in various media.

Synthesis and sweetness characteristics of L-aspartyl-D-alanine fenchyl esters.

Four isomers of the L-aspartyl-D-alanine fenchyl esters were prepared as potential peptide sweeteners. L-Aspartyl-D-alanine (+)-alpha-fenchyl ester and L-aspartyl-D-alanine (-)-beta-fenchyl ester showed sweetness with potencies 250 and 160 times higher than that of sucrose, respectively. In contrast, L-aspartyl-D-alanine (+)-beta-fenchyl ester and L-aspartyl-D-alanine (-)-alpha-fenchyl ester had the highest sweetness potencies at 5700 and 1100 times that of sucrose, respectively. In particular, L-aspartyl-D-alanine (-)-alpha-fenchyl ester had an excellent sweetness quality; but L-aspartyl-D-alanine (+)-beta-fenchyl ester did not have an excellent quality of sweetness because it displayed an aftertaste caused by the strong sweetness.

Analysis of rapid, large-scale protein quaternary structural changes: time-resolved X-ray solution scattering of Nudaurelia capensis omega virus (NomegaV) maturation.

Time-resolved small-angle X-ray scattering (TR-SAXS) was used to study the kinetics of a large conformational change that occurs during the maturation of an icosahedral virus. Virus-like particles (VLPs) of the T=4 non-enveloped RNA virus Nudaurelia capensis omega virus (NomegaV) were shown to undergo a large pH-dependent conformational change. Electron cryo-microscopy (cryoEM) and X-ray solution scattering were used to show that the precursor VLP (procapsid) was 16 % larger in diameter than the resulting capsid, which was shown by the cryoEM study to closely resemble the infectious mature virion. The procapsid form of the VLPs was observed at pH 7.5 and was converted to the capsid form at pH 5.0. Static SAXS measurements of the VLPs in solutions ranging between these pH values determined that the half-titration point of the transition was pH 6.0. Time-resolved SAXS experiments were performed on VLP solutions by initiating a pH change from 7.5 to 5.0 using a stopped-flow device, and the time-scale of the conformational change occurred in the subsecond range. Using a less drastic pH change (lowering the pH to 5.8 or 5.5), the conformational change occurred more slowly, on the subminute or minute time-scale, with the detection of a fast-forming intermediate in the transition. Further characterization using static SAXS measurements showed that the conformational change was initially reversible but became irreversible after autoproteolytic maturation was about 15 % complete. In addition to characterizing the large quaternary conformational change, we have been able for the first time to demonstrate that it takes place on the subsecond time-scale, a regime comparable to that observed in other multisubunit assemblies.

Structure and mechanism of the RuvB Holliday junction branch migration motor.

The RuvB hexamer is the chemomechanical motor of the RuvAB complex that migrates Holliday junction branch-points in DNA recombination and the rescue of stalled DNA replication forks. The 1.6 A crystal structure of Thermotoga maritima RuvB together with five mutant structures reveal that RuvB is an ATPase-associated with diverse cellular activities (AAA+-class ATPase) with a winged-helix DNA-binding domain. The RuvB-ADP complex structure and mutagenesis suggest how AAA+-class ATPases couple nucleotide binding and hydrolysis to interdomain conformational changes and asymmetry within the RuvB hexamer implied by the crystallographic packing and small-angle X-ray scattering in solution. ATP-driven domain motion is positioned to move double-stranded DNA through the hexamer and drive conformational changes between subunits by altering the complementary hydrophilic protein- protein interfaces. Structural and biochemical analysis of five motifs in the protein suggest that ATP binding is a strained conformation recognized both by sensors and the Walker motifs and that intersubunit activation occurs by an arginine finger motif reminiscent of the GTPase-activating proteins. Taken together, these results provide insights into how RuvB functions as a motor for branch migration of Holliday junctions.

Fourier amplitude decay of electron cryomicroscopic images of single particles and effects on structure determination.

Several factors, including spatial and temporal coherence of the electron microscope, specimen movement, recording medium, and scanner optics, contribute to the decay of the measured Fourier amplitude in electron image intensities. We approximate the combination of these factors as a single Gaussian envelope function, the width of which is described by a single experimental B-factor. We present an improved method for estimating this B-factor from individual micrographs by combining the use of X-ray solution scattering and numerical fitting to the average power spectrum of particle images. A statistical estimation from over 200 micrographs of herpes simplex virus type-1 capsids was used to estimate the spread in the experimental B-factor of the data set. The B-factor is experimentally shown to be dependent on the objective lens defocus setting of the microscope. The average B-factor, the X-ray scattering intensity of the specimen, and the number of particles required to determine the structure at a lower resolution can be used to estimate the minimum fold increase in the number of particles that would be required to extend a single particle reconstruction to a specified higher resolution. We conclude that microscope and imaging improvements to reduce the experimental B-factor will be critical for obtaining an atomic resolution structure.

Domain bridging interactions. A necessary contribution to the function and structure of Escherichia coli aspartate transcarbamoylase.

Aspartate transcarbamoylase undergoes a domain closure in the catalytic chains upon binding of the substrates that initiates the allosteric transition. Interdomain bridging interactions between Glu(50) and both Arg(167) and Arg(234) have been shown to be critical for stabilization of the R state. A hybrid version of the enzyme has been generated in vitro containing one wild-type catalytic subunit, one catalytic subunit in which Glu(50) in each catalytic chain has been replaced by Ala (E50A), and wild-type regulatory subunits. Thus, the hybrid enzyme has one catalytic subunit capable of domain closure and one catalytic subunit incapable of domain closure. The hybrid does not behave as a simple mixture of the constituent subunits; it exhibits lower catalytic activity and higher aspartate affinity than would be expected. As opposed to the wild-type enzyme, the hybrid is inhibited allosterically by CTP at saturating substrate concentrations. As opposed to the E50A holoenzyme, the hybrid is not allosterically activated by ATP at saturating substrate concentrations. Small angle x-ray scattering showed that three of the six interdomain bridging interactions in the hybrid is sufficient to cause the global structural change to the R state, establishing the critical nature of these interactions for the allosteric transition of aspartate transcarbamoylase.

Direct structural evidence for a concerted allosteric transition in Escherichia coli aspartate transcarbamoylase.

Regulation of protein function, often achieved by allosteric mechanisms, is central to normal physiology and cellular processes. Although numerous models have been proposed to account for the cooperative binding of ligands to allosteric proteins and enzymes, direct structural support has been lacking. Here, we used a combination of X-ray crystallography and small angle X-ray scattering in solution to provide direct structural evidence that the binding of ligand to just one of the six active sites of Escherichia coli aspartate transcarbamoylase induces a concerted structural transition from the T to the R state.

Characterization of subunit structural changes accompanying assembly of the bacteriophage P22 procapsid.

P22 serves as a model for the assembly and maturation of icosahedral double-stranded DNA viruses. The viral capsid precursor, or procapsid, is assembled from 420 copies of a 47 kDa coat protein subunit (gp5) that is rich in beta-strand secondary structure. Maturation to the capsid, which occurs in vivo upon DNA packaging, is accompanied by shell expansion and a large increase in the level of protection against deuterium exchange of amide NH groups. Accordingly, shell maturation resembles the final step in protein folding, wherein domain packing and an exchange-protected core become more fully developed [Tuma, R., Prevelige, P. E., Jr., and Thomas, G. J., Jr. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9885-9890]. Here, we exploit recent advances in Raman spectroscopy to investigate the P22 coat protein subunit under conditions which stabilize the monomeric state, viz., in solution at very low concentrations. Under these conditions, the monomer exhibits an elongated shape, as demonstrated by small-angle X-ray scattering. Raman spectra allow the identification of conformation-sensitive marker bands of the monomer, as well as the characterization of NH exchange dynamics for comparison with procapsid and capsid shell assemblies. We show that procapsid assembly involves significant ordering of the predominantly beta-strand backbone. We propose that such ordering may mediate formation of the distinct subunit conformations required for assembly of a T = 7 icosahedral lattice. However, the monomer, like the subunit within the procapsid lattice, exhibits a moderate level of protection against low-temperature NH exchange, indicative of a nascent folding core. The environments and exchange characteristics of key side chains are also similar for the monomeric and procapsid subunits, and distinct from corresponding characteristics of the capsid subunit. The monomer thus represents a compact but metastable folding intermediate along the pathway to assembly of the procapsid and capsid.

Crystal and solution structures of an HslUV protease-chaperone complex.

HslUV is a "prokaryotic proteasome" composed of the HslV protease and the HslU ATPase, a chaperone of the Clp/Hsp100 family. The 3.4 A crystal structure of an HslUV complex is presented here. Two hexameric ATP binding rings of HslU bind intimately to opposite sides of the HslV protease; the HslU "intermediate domains" extend outward from the complex. The solution structure of HslUV, derived from small angle X-ray scattering data under conditions where the complex is assembled and active, agrees with this crystallographic structure. When the complex forms, the carboxy-terminal helices of HslU distend and bind between subunits of HslV, and the apical helices of HslV shift substantially, transmitting a conformational change to the active site region of the protease.

The effect of a hemofilter during extracorporeal circulation on hemodynamics in patients with SIRS.

OBJECTIVE: To evaluate the hypothesis that extracorporeal hemopurification with a hemofilter contributes to the stabilization of hemodynamics in patients with systemic inflammatory response syndrome (SIRS) due to a mechanism other than the removal of cytokines. DESIGN: Prospective study. SETTING: Intensive care unit (ICU) in a university hospital. PATIENTS: Seven critically ill patients who met criteria for SIRS with unstable hemodynamics requiring vasopressors after emergency surgery. INTERVENTIONS: Before initiation of continuous hemofiltration (CHF) extracorporeal circulation through the hemofilter (ECC) with a hollow-fiber polyacrylonitrile hemofilter was performed for 6 h. MEASUREMENTS AND RESULTS: Vital signs were monitored continuously and hemodynamics were evaluated intermittently. The blood level of endotoxin and the plasma levels of cytokines were measured at 0 and 6 h. Changes in plasma levels of cytokines passing through the hemofilter were evaluated at 3 h. A significant decrease of body temperature (P < 0.05 at 3 and 6 h vs 0 h), a significant elevation of mean arterial pressure (P < 0.05 at 0.5 h, P < 0.01 at 3 and 6 h vs 0 h), and a significant increase of urinary flow rate (P < 0.05 at 0 to 3 h vs -3 to 0 h) were observed with ECC. Neither the blood level of endotoxin nor the plasma levels of cytokines decreased. A significant increase of plasma IL-6 as it passed through the hemofilter was noted. CONCLUSIONS: The beneficial effects of hemopurification with a hemofilter on SIRS patients are possibly due to mechanisms other than the elimination of cytokines with ultrafiltration, diffusion, or adsorption.