Plasma Proteomic Analysis Reveals the Potential Role of Lectin and Alternative Complement Pathways in IgA Vasculitis Pathogenesis.

BACKGROUND: IgA vasculitis (IgAV) is the most common form of childhood vasculitis. A better understanding of its pathophysiology is required to identify new potential biomarkers and treatment targets. OBJECTIVE: to assess the underlying molecular mechanisms in the pathogenesis of IgAV using an untargeted proteomics approach. METHODS: Thirty-seven IgAV patients and five healthy controls were enrolled. Plasma samples were collected on the day of diagnosis before any treatment was initiated. We used nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) to investigate the alterations in plasma proteomic profiles. For the bioinformatics analyses, databases including Uniprot, PANTHER, KEGG, Reactome, Cytoscape, and IntAct were used. RESULTS: Among the 418 proteins identified in the nLC-MS/MS analysis, 20 had significantly different expressions in IgAV patients. Among them, 15 were upregulated and 5 were downregulated. According to the KEGG pathway and function classification analysis, complement and coagulation cascades were the most enriched pathways. GO analyses showed that the differentially expressed proteins were mainly involved in defense/immunity proteins and the metabolite interconversion enzyme family. We also investigated molecular interactions in the identified 20 proteins of IgAV patients. We extracted 493 interactions from the IntAct database for the 20 proteins and used Cytoscape for the network analyses. CONCLUSION: Our results clearly suggest the role of the lectin and alternate complement pathways in IgAV. The proteins defined in the pathways of cell adhesion may serve as biomarkers. Further functional studies may lead the way to better understanding of the disease and new therapeutic options for IgAV treatment.

Immobilization of Candida rugosa lipase on magnetic chitosan beads and application in flavor esters synthesis.

In this work, magnetic chitosan (MCH) beads were synthesized by phase-inversion method, and grafted with polydopamine (PDA) and then used for direct immobilization of Candida rugosa lipase by Schiff base reaction. The amount of immobilized enzyme and the retained activity were found to be 47.3 mg/g and 72.8%, respectively, at pH 7.0, and at 25 °C. The apparent Km (9.7 mmol/L), and Vmax (384 U/mg) values of the immobilized lipase were significantly changed compared to the free lipase. The MCH@PDA-lipase was better thermal and storage stability at different temperatures than those of the free lipase. In hexane medium, the esterification reaction results showed that the maximum conversions of isoamylalcohol and isopentyl alcohol to isoamyl acetate and isopentyl acetate using the MCH@PDA-lipase were found to be 98.4 ± 1.3% and 73.7 ± 0.7%, respectively. These results showed that the MCH@PDA-lipase can be used as an operative immobilized enzyme system for many biotechnological applications.

Following hybridization on sensor/array platforms by using SPR, elipsometer and MALDI-MS.

The aim of this study is to develop a methodology in which Surface Plasmon Resonance (SPR), Ellipsometer (EM) and Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry (MALDI-MS) will be used together for detection of single-strand oligodeoxynucleotides (ssODNs) targets. A selected target-ssODNs, and its complementary, the probe-ssODNs carrying a -SH end group, a spacer arm (HS-(CH2)6-(T)15, and a non-complementary ssODNs were used. Silicone based stamps with 16 regions were prepared and used for micro-contact printing (µCP) of the probe-ssODNs on the gold coated surfaces homogeneously. A modulator-spacer molecule (6-mercapto-1-hexanol) was co-immobilized to control surface probe density, to orientate the probe-ssODNs, and to eliminate the nonspecific interactions. SPR was used successfully to follow the hybridization of the target-ssODNs with the immobilized probe-ssODNs on the platform surfaces. Complete hybridizations were achieved in 100 min. It was obtained that there was a linear relationship between relative change in delta and target concentration below 1 µm. Using imaging version of ellipsometer (IEM) allowed imaging of the surfaces and supported extra datum for the SPR results. After a very simple dehybridization protocol, MALDI-MS analysis allowed detection of the target-ssODNs hybridized on the sensor/array platforms.

Ti (IV) attached-phosphonic acid functionalized capillary monolith as a stationary phase for in-syringe-type fast and robust enrichment of phosphopeptides.

In this study, poly(vinylphosphonic acid-co-ethylene dimethacrylate), poly(VPA-co-EDMA) capillary monolith was synthesized as a starting material for obtaining a stationary phase for microscale enrichment of phosphopeptides. The chelation of active phosphonate groups with Ti (IV) ions gave a macroporous monolithic column with a mean pore size of 5.4 µm. The phosphopeptides from different sources were enriched on Ti (IV)-attached poly(VPA-co-EDMA) monolith using a syringe-pump. The monolithic capillary columns exhibited highly sensitive/selective enrichment performance with phosphoprotein concentrations as low as 1.0 fmol/mL. Six different phosphopeptides were detected with high intensity by the treatment of ß-casein digest with the concentration of 1.0 fmol/mL, using Ti (IV)@poly(VPA-co-EDMA) monolith. Highly selective enrichment of phosphopeptides was also successfully carried out even at trace amounts, in a complex mixture of digested proteins (molar ratio of ß-casein to bovine serum albumin, 1:1500) and three phosphopeptides were successfully detected. Four highly intense signals of phosphopeptides in human serum were also observed with high signal-to-noise ratio and a clear background after enrichment with Ti (IV)@poly(VPA-co-EDMA) monolith. It was concluded that the capillary microextraction system enabled fast, efficient and robust enrichment of phosphopeptides from microscale complex samples. The whole enrichment process was completed within 20 min, which was shorter than in the previously reported studies.

Ti(IV) carrying polydopamine-coated, monodisperse-porous SiO2 microspheres with stable magnetic properties for highly selective enrichment of phosphopeptides.

A marked decrease in the saturation magnetization by the formation of functional shells around the magnetic core is an important disadvantage of magnetic core-shell nanoparticles. Another drawback of Ti(IV)-functionalized immobilized metal affinity chromatography (IMAC) sorbents is the acidic character of the binding medium used for Ti4+ attachment onto composite magnetic nanoparticles, which causes an additional decrease in the saturation magnetization owing to the chemical interaction between the acidic moiety and the magnetic core. An IMAC sorbent in the form of magnetic microspheres with superior and stable magnetic properties with respect to magnetic core-shell nanoparticles was designed for phosphopeptide enrichment. Magnetic, monodisperse-porous silica microspheres (MagSiO2) 6µm in size were synthesized by a new staged-shape template hydrolysis-condensation protocol. A porous-silica shell layer was generated around the microspheres to protect the magnetic core from the acidic medium during Ti4+ attachment (MagSiO2@SiO2). The MagSiO2@SiO2 microspheres were coated with a polydopamine shell (MagSiO2@SiO2@PDA) and Ti4+ was attached onto the composite microspheres (MagSiO2@SiO2@PDA@Ti(IV)). Formation of the PDA layer and Ti4+ attachment did not cause any significant decrease in the saturation magnetization. The platform exhibited excellent performance for phosphopeptide enrichment from the digests of phosphorylated proteins. Selectivity was investigated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The detection limit for phosphopeptide enrichment by the MagSiO2@SiO2@PDA@Ti(IV) microspheres from the tryptic digests of ß-casein was 50 fmol/mL. Usability of the proposed magnetic sorbent with complex biological samples was demonstrated by successful enrichment of four phosphopeptides from human serum. The proposed sorbent showed stable performance over five repeated uses.

Highly selective enrichment of phosphopeptides by titanium (IV) attached monodisperse-porous poly(vinylphosphonic acid-co-ethylene dimethacrylate) microspheres.

A seeded polymerization protocol was developed for the synthesis of monodisperse-porous poly(vinylphosphonic acid-co-ethylene dimethacrylate), [poly(VPA-co-EDMA)] microspheres with superior porous properties. The protocol allowed the direct synthesis of phosphonic acid functionalized porous microspheres with the mean size of ∼4µm and the specific surface area of 420m2g-1 without applying any complicated post-derivatization protocol for the attachment of phosphonic acid group. The phosphonic acid content of poly(VPA-co-EDMA) microspheres was determined as 1.5mmol H2PO3g-1 microspheres. Ti(IV) ions were attached onto the microspheres via metal-chelate complex formation by phosphonate-groups and Ti(IV) carrying monodisperse-porous poly(vinylphosphonic acid-co-ethylene dimethacrylate), [Ti(IV)@poly(VPA-co-EDMA)] microspheres were obtained as a new sorbent for phosphopeptide enrichment via immobilized metal affinity chromatography. The phosphopeptides in the enriched samples were identified by matrix-assited laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Four different phosphopeptides were detected with extremely high intensity by the treatment of ß-casein digest prepared with the concentration of 10 fmol/mL with Ti(IV)@poly(VPA-co-EDMA) microspheres. Highly selective enrichment of phosphopeptides was also successfully carried out even at trace amounts in a complex mixture of digested proteins (molar ratio of ß-casein to BSA, 1:1000) and eight different phosphorylated peptides from BSA digest were successfully identified. Moreover, four highly intense signals of the phosphopeptides in human serum were observed with high S/N ratio and clear background after enrichment by using Ti(IV)@poly(VPA-co-EDMA) microspheres.

Monitoring protein glycation by electrospray ionization (ESI) quadrupole time-of-flight (Q-TOF) mass spectrometer.

In this study electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) mass spectrometry was used to investigate protein glycation. The glycated species of cytochrome C, lysozyme, and ß-casein formed during glycation with d-glucose were identified and monitored in binary systems heated at 70°C under dry and aqueous conditions. Cytochrome C had multiple charges in non-glycated state, primarily changing from +13 to +17 positive charges, whereas ß-casein had charge states up to +30. Upon heating with glucose at 70°C in aqueous state, attachment of one glucose molecule onto proteins was observed in each charge state. However, heating in dry state caused much more glucose attachment, leading to the formation of multiple glycoforms of proteins. By using ESI-QTOF-MS technique, formation of glycated cytochrome C containing up to 12 glucose moieties were observed, while glycated species containing 6 and 8 glucose moieties were observed for lysozyme and ß-casein, respectively in various heating conditions.

Removal of bisphenol A from aqueous medium using molecularly surface imprinted microbeads.

The aim of this study is to prepare bisphenol A (BPA) imprinted polymers, which can be used for the selective removal of BPA from aqueous medium. The BPA-imprinted (MIP) and non-imprinted (NIP) microbeads were synthesized, and characterized by Zeta-sizer, FTIR, SEM and BET method. Bisphenol A was determined in solutions using liquid chromatography-mass spectroscopy (LC-MS). The effect of initial concentration of BPA, the adsorption rate and the pH of the medium on the capacity of BPA-imprinting polymer were studied. Adsorption capacity of BPA was affected by the amount of the incorporated functional monomer in the polymer network. BPA adsorption capacity of MIP-3 and NIP microbeads from aqueous medium was estimated as 76.7 and 59.9 mg g(-1), respectively. The binding efficiencies of BPA-MIP-3 microbeads for different phenolic compounds (i.e., BPA with p-toluidine, 4-aminophenol or 2-naphthol) were explored at binary solutions, and the binding capacities of BPA-imprinted microbeads were found to be 2.79 × 10(-1), 2.39 × 10(-1), 7.59 × 10(-2) and 5.48 × 10(-2) mmol g(-1) microbeads, respectively. The satisfactory results demonstrated that the obtained BPA-MIP microbeads showed an appreciable binding specificity toward BPA than similar structural compounds in the aqueous medium. Moreover, the reusability of BPA-MIP-3 microbeads was tested for several times and no significant loss in adsorption capacity was observed. Finally, the binary and multi-component systems results show that MIP-3 microbeads have special recognition selectivity and excellent binding affinity for template molecule "BPA".

Comparison of two methods for purification of enterocin B, a bacteriocin produced by Enterococcus faecium W3.

This study aimed to compare two different approaches for the purification of enterocin B from Enterococcus faecium strain W3 based on the observation that the bacteriocin was found both in cell associated form and in culture supernatant. The first approach employed ammonium sulfate precipitation, cation-exchange chromatography, and sequential reverse-phase high-performance liquid chromatography. The latter approach exploited a pH-mediated cell adsorption-desorption method to extract cell-bound bacteriocin, and one run of reverse-phase chromatography. The first method resulted in purification of enterocin B with a recovery of 4% of the initial bacteriocin activity found in culture supernatant. MALDI-TOF MS analysis and de novo peptide sequencing of the purified bacteriocin confirmed that the active peptide was enterocin B. The second method achieved the purification of enterocin B with a higher recovery (16%) and enabled us to achieve pure bacteriocin within a shorter period of time by avoiding time consuming purification protocols. The purity and identity of the active peptide were confirmed again by matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry (MS) analysis. Although both approaches were satisfactory to obtain a sufficient amount of enterocin B for use in MS and amino acid sequence analysis, the latter was proved to be applicable in large-scale and rapid purification of enterocin B.

Strong ionic interactions in noncovalent complexes between poly(ethylene imine), a cationic electrolyte, and Cibacron Blue, a nucleotide mimic--implications for oligonucleotide vectors.

Cationic polymers can bind DNA to form polyplexes, which are noncovalent complexes used for gene delivery into the targeted cells. For more insight on such biologically relevant systems, the noncovalent complexes between the cationic polymer poly(ethylene imine) (PEI) and the nucleotide mimicking dye Cibacron Blue F3G-A (CB) were investigated using mass spectrometry methods. Two PEIs of low molecular weight were utilized (Mn ≈ 423 and 600 Da). The different types of CB anions produced by Na(+)/H(+) exchanges on the three sulfonic acid groups of CB and their dehydrated counterparts were responsible for complex formation with PEI. The CB anions underwent noncovalent complex formation with protonated, but not with sodiated PEI. A higher proportion of cyclic oligomers were detected in PEI423 than PEI600, but both architectures formed association products with CB. Tandem mass spectrometry studies revealed a significantly stronger noncovalent interaction between PEI and dehydrated CB than between PEI and intact CB.

Effects of 900 MHz radiofrequency radiation on skin hydroxyproline contents.

The present study aimed to investigate the possible effect of pulse-modulated radiofrequency radiation (RFR) on rat skin hydroxyproline content, since skin is the first target of external electromagnetic fields. Skin hydroxyproline content was measured using liquid chromatography mass spectrometer method. Two months old male wistar rats were exposed to a 900 MHz pulse-modulated RFR at an average whole body specific absorption rate (SAR) of 1.35 W/kg for 20 min/day for 3 weeks. The radiofrequency (RF) signals were pulse modulated by rectangular pulses with a repetition frequency of 217 Hz and a duty cycle of 1:8 (pulse width 0.576 ms). A skin biopsy was taken at the upper part of the abdominal costa after the exposure. The data indicated that whole body exposure to a pulse-modulated RF radiation that is similar to that emitted by the global system for mobile communications (GSM) mobile phones caused a statistically significant increase in the skin hydroxyproline level (p = 0.049, Mann-Whitney U test). Under our experimental conditions, at a SAR less than the International Commission on Non-Ionizing Radiation Protection safety limit recommendation, there was evidence that GSM signals could alter hydroxyproline concentration in the rat skin.

Specific enrichment and direct detection of phosphopeptides on insoluble transition metal oxide particles in matrix-assisted laser desorption/ionization mass spectrometry applications.

Several transition metal oxides, such as iron (III), nickel (II) and zirconium (IV) oxides, were examined in detail for the specific enrichment and the purification of phosphopeptides from a digested peptide mixture solution. Phosphopeptide enrichment was performed on the metal oxide particles using a peptide mixture obtained bytryptic digestion of beta-casein. The mixture of protein digests containing bovine serum albumin (BSA): beta-casein digests (100:1 mole ratio) was also used for the phosphopeptide enrichment. Furthermore, non-fat milk digest was examined as a complex biological sample. In each phosphopeptide enrichment process, phosphopeptides were specifically enriched and separated from the non-phosphopeptides. The phosphopeptides were adsorbed onto the metal oxide surface at acidic pH values between 1.0 and 2.0 and, for desorption of phosphopeptides from metal oxide particles, pH values were examined and optimized in the enrichment studies. The analysis of phosphopeptides were carried out by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) using 2,5-dihydroxybenzoic acid matrix containing 1.0% phosphoric acid to obtain intense protonated signals and to overcome degradation of the phosphopeptides by phosphate group loss in mass spectrometric conditions. Moreover, it was demonstrated that the direct detection of phosphopeptides from the surface of the metal oxide particles was possible using MALDI-MS by mixing the phosphopeptide-adsorbed metal oxide particles with MALDI matrix solution in slurry form before the analysis. Thus, the effects of interferences arising from chemical species used in the desorption process was successfully eliminated for the fast and sensitive detection of phosphopeptides in MALDI-MS applications.

A novel tantalum-based sol-gel packed microextraction syringe for highly specific enrichment of phosphopeptides in MALDI-MS applications.

A new tantalum-based sol-gel material was synthesized using a unique sol-gel synthesis pathway by PEG incorporation into the sol-gel structure without performing a calcination step. This improved its chemical and physical properties for the high capacity and selective enrichment of phosphopeptides from protein digests in complex biological media. The specificity of the tantalum-based sol-gel material for phosphopeptides was evaluated and compared with tantalum(V) oxide (Ta2O5) in different phosphopeptide enrichment applications. The tantalum-based sol-gel and tantalum(V) oxide were characterized in detail using FT-IR spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM), and also using a surface area and pore size analyzer. In the characterization studies, the surface morphology, pore volume, crystallinity of the materials and PEG incorporation into the sol-gel structure to produce a more hydrophilic material were successfully demonstrated. The X-ray diffractograms of the two different materials were compared and it was noted that the broad signals of the tantalum-based sol-gel clearly represented the amorphous structure of the sol-gel material, which was more likely to create enough surface area and to provide more accessible tantalum atoms for phosphopeptides to be easily adsorbed when compared with the neat and more crystalline structure of Ta2O5. Therefore, the phosphopeptide enrichment performance of the tantalum-based sol-gels was found to be remarkably higher than the more crystalline Ta2O5 in our studies. Phosphopeptides at femtomole levels could be selectively enriched using the tantalum-based sol-gel and detected with a higher signal-to-noise ratio by matrix-assisted laser desorption/ionization-mass spectrometer (MALDI-MS). Moreover, phosphopeptides in a tryptic digest of non-fat bovine milk as a complex real-world biological sample were retained with higher yield using a tantalum-based sol-gel. Additionally, the sol-gel material was packed into a standard syringe (0.5 mL) to enhance the ease of use of the sol-gel material and for the elimination of additional mixing and separation procedures during the adsorption, washing and elution steps of the enrichment procedure. It was found that up to 28 phosphopeptides in milk digest were easily detectable by MALDI-MS at femtomole levels (around 20 fmol) using the microextraction syringe within less than one minute.

Small molecule analysis using laser desorption/ionization mass spectrometry on nano-coated silicon with self-assembled monolayers.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an emerging technique for the determination of the molecular weight of biomolecules and their non-covalent complexes without fragmentation. One problem with this technique is the use of excess amounts of matrices, which may produce intense fragment ions and/or clusters at low mass ranges between 1 and 800 Da. These fragments lead to interference, especially concerning the signals of small target molecules. Here, a simple, reusable, and quite inexpensive approach was demonstrated to improve the effectiveness of laser desorption/ionization mass spectrometry (LDI-MS) analysis, especially for small molecules, without using matrix molecules. In this study, substrates with controllable morphologies and thicknesses were developed based on the self-assembly of silane molecules on silicon surfaces using N-(3-trimethoxysilylpropyl)diethylenetriamine (TPDA) and octadecyltrichlorosilane (OTS) molecules. Prepared substrates with nano-overlayers were successfully used in the analysis of different types of small target molecules, namely acrivastine, L-histidine, L-valine, L-phenylalanine, L-arginine, L-methionine and angiotensin I. Our substrates exhibited clear peaks almost without fragmentation for all target molecules, suggesting that these surfaces provide a number of important advantages for LDI-MS analysis, such as ease of preparation, costs, reusability, robustness, easy handling and preventing fragmentation.

Amine-functionalized sol-gel-based lab-in-a-pipet-tip approach for the fast enrichment and specific purification of phosphopeptides in MALDI-MS applications.

Amine-functionalized sol-gels were investigated for the enrichment and purification of phosphopeptides from digested protein mixture solutions. Tetramethylorthosilicate (TMOS) and N'[3-(trimethoxysilyl)-propyl]-diethylenetriamine (TPDA) were used in a 1:1 mole ratio in the production of amine-functionalized sol-gels. The sol-gel network was then used for phosphopeptide enrichment. Phosphopeptide enrichment onto the synthesized amine-functionalized sol-gels was performed using an enolase digested peptide mixture, a ß-casein digested peptide mixture, as well as these digested peptide mixtures contaminated 50-fold with bovine serum albumin (BSA). Moreover, phosphopeptide enrichment was successfully performed using nonfat milk as a highly contaminated and complex material. In each phosphopeptide enrichment and purification process, only phosphopeptides were enriched and separated from the other digested peptides. Phosphopeptides were adsorbed onto the amine-functionalized sol-gels at pH 4.0 and eluted at pH 1.0 using trifluoroacetic acid (TFA). For phosphopeptide analysis by MALDI-MS, a 2,5-dihydroxybenzoic acid matrix containing 1.0% phosphoric acid was used to overcome the degradation of phosphopeptides and provide high intensity phosphopeptide protonated molecular ion signal intensities. It was also found that phosphopeptide detection limits were improved to approximately 10 femtomoles. For rapid and specific phosphopeptide enrichment and purification, sol-gel materials were placed in a 10 µL pipet tip with glass wool on either side. Phosphopeptide enrichment from digested peptide mixtures was performed in a very short time (less than 1 min) at subpicomole levels using this novel lab-in-a-pipet-tip approach.

Novel short peptides isolated from phage display library inhibit vascular endothelial growth factor activity.

Signal transduction through the vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR) pathway has a pivotal importance in angiogenesis, and has therefore become a prime target in antitumor therapy. In search for peptides antagonizing VEGF binding to its receptors, we screened a random heptamer library displayed on phage for peptides that bind the whole VEGF165 molecule and inhibit VEGF dependent human umbilical vein endothelial cell (HUVEC) proliferation. Two selected peptides with sequences WHLPFKC and WHKPFRF were synthesized. Biacore and matrix-assisted laser desorption/ionization timeof- flight mass spectrometry analysis indicated that these peptides bind the VEGF homodimer in a concentration- dependent manner, with micromolar affinity, and with a 2:1 peptide:VEGF stoichiometry. They inhibited HUVEC proliferation in vitro by 77 and 55%, respectively. Taken together, our results indicate that these peptides could be potent inhibitors of angiogenesis. Furthermore, we show that the peptide- VEGF binding properties can be quantified, a prerequisite for the further optimization of binders.

Matrix elimination method for the determination of precious metals in ores using electrothermal atomic absorption spectrometry.

Poly(N-(hydroxymethyl)methacrylamide)-1-allyl-2-thiourea) hydrogels, poly(NHMMA-ATU), were synthesized by gamma radiation using (60)Co gamma source in the ternary mixture of NHMMA-ATU-H(2)O. These hydrogels were used for the specific gold, silver, platinum and palladium recovery, pre-concentration and matrix elimination from the solutions containing trace amounts of precious metal ions. Elimination of inorganic matrices such as different transition and heavy metal ions, and anions was performed by adjusting the solution pH to 0.5 that was the selective adsorption pH of the precious metal ions. Desorption of the precious metal ions was performed by using 0.8 M thiourea in 3M HCl as the most efficient desorbing agent with recovery values more than 95%. In the desorption medium, thiourea effect on the atomic signal was eliminated by selecting proper pyrolysis and atomization temperatures for all precious metal ions. Precision and the accuracy of the results were improved in the graphite furnace-atomic absorption spectrometer (GFAAS) measurements by applying the developed matrix elimination method performing the adsorption at pH 0.5. Pre-concentration factors of the studied precious metal ions were found to be at least 1000-fold. Detection limits of the precious metal ions were found to be less than 10 ng L(-1) of the all studied precious metal ions by using the proposed pre-concentration method. Determination of trace levels of the precious metals in the sea-water, anode slime, geological samples and photographic fixer solutions were performed using GFAAS clearly after applying the adsorption-desorption cycle onto the poly(NHMMA-UTU) hydrogels.