Immobilization of urease on nanostructured polymer membrane and preparation of urea amperometric biosensor

A new matrix for enzyme immobilization of urease was obtained by incorporating rhodium nanoparticles (5% on activated charcoal) and chemical bonding of chitosan with different concentration (0.15%; 0.3%; 0.5%; 1.0%; 1.5%) in previously chemically modified AN copolymer membrane. The basic characteristics of the chitosan modified membranes were investigated. The SEM analyses were shown essential morphology change in the different modified membranes. Both the amount of bound protein and relative activity of immobilized enzyme were measured. A higher activity (about 77.44%) was measured for urease bound to AN copolymer membrane coated with 1.0% chitosan and containing rhodium nanoparticles. The basic characteristics (pH(opt), T(opt), thermal, storage and operation stability) of immobilized enzyme on this optimized modified membrane were also determined. The prepared enzyme membrane was used for the construction of amperometric biosensor for urea detection. Its basic amperometric characteristics were investigated. A calibration plot was obtained for urea concentration ranging from 1.6 to 23 mM. A linear interval was detected along the calibration curve from 1.6 to 8.2mM. The sensitivity of the constructed biosensor was calculated to be 3.1927 μAmM(-1)cm(-2). The correlation coefficient for this concentration range was 0.998. The detection limit with regard to urea was calculated to be 0.5mM at a signal-to-noise ratio of 3. The biosensor was employed for 10 days while the maximum response to urea retained 86.8%.     

Integrated constructed wetland systems employing alum sludge and oyster shells as filter media for P removal

This research aimed to investigate the technical feasibility of integrated constructed wetland system consisting of a pre-filter unit and a constructed wetland (CW), in series; packed with alum sludge (AS) and oyster shells (OS) as the filter media, respectively, for nitrogen and phosphorus removal from domestic wastewater. Based on the 240 days of operation from January to August 2007, this integrated system was highly effective in removing BOD, N, P and TSS compounds which were found to be 89.5%, 68.8%, 99.4% and 89.9%, respectively. After this period, the integrated system was modified as the CW and post-filter unit, in series. The post-filter of this modified integrated system was operated during 60 days with cover for light shield and during another 60 days with no cover from September to December 2007. The treatment performance of modified integrated system was effective in removing BOD₅, N, P and TSS compounds which were found to be 91.4%, 86.8%, 99.7% and 73%, respectively, during which the post-filter had operated with no cover. To simulate high rainfall conditions, the integrated system was tested under hydraulic shock loading at the overall hydraulic retention time of 0.7 day during one day. This hydraulic shock loading conditions made BOD₅, TN, TSS concentration increase, but made no effect on P concentration. Integrated system combined a pre-filter and a CW unit or a CW unit followed by a post-filter is recommended for use in domestic wastewater which should result in high treatment performance, especially on P removal.     

A novel urine analysis technique combining affinity chromatography with Au nanoparticle based surface enhanced Raman spectroscopy for potential applications in non‐invasive cancer screening

Modified nucleoside in urine samples is one of the most common biomarkers for cancer screening. Therefore, we developed a novel detection method for modified nucleoside detection in human urine. In this work, the modified nucleoside from real cancer patient's urine samples was first separated and purified using the affinity chromatography (AC) technology relying on its specific adsorption capacity. Then, surface‐enhanced Raman spectroscopy (SERS) technology with the capability of single molecular detection was used to sensitively characterize the biomolecular features of modified nucleoside. A total of 141 high‐quality SERS spectra of urinary modified nucleoside can be obtained from 50 gastric cancer patients and 43 breast cancer patients, as well as 48 healthy volunteers. Using principal component analysis combined with linear discriminant analysis (PCA‐LDA), the diagnostic sensitivities for identifying gastric cancer vs normal, breast cancer vs normal, gastric cancer vs breast cancer were 84.0%, 76.7% and 82.0%, respectively, and the corresponding diagnostic specificities for each combination were 95.8%, 87.5% and 90.7%, respectively. These results show that this novel method based on urinary modified nucleoside detection combining AC and SERS technologies holds promising potential for developing a specific, non‐invasive and label‐free tool for cancer screening.      

Using Mathematical Algorithms to Modify Glomerular Filtration Rate Estimation Equations

Background

The equations provide a rapid and low-cost method of evaluating glomerular filtration rate (GFR). Previous studies indicated that the Modification of Diet in Renal Disease (MDRD), Chronic Kidney Disease-Epidemiology (CKD-EPI) and MacIsaac equations need further modification for application in Chinese population. Thus, this study was designed to modify the three equations, and compare the diagnostic accuracy of the equations modified before and after.

Methodology

With the use of ^{99 m}Tc-DTPA renal dynamic imaging as the reference GFR (rGFR), the MDRD, CKD-EPI and MacIsaac equations were modified by two mathematical algorithms: the hill-climbing and the simulated-annealing algorithms.

Results

A total of 703 Chinese subjects were recruited, with the average rGFR 77.14±25.93 ml/min. The entire modification process was based on a random sample of 80% of subjects in each GFR level as a training sample set, the rest of 20% of subjects as a validation sample set. After modification, the three equations performed significant improvement in slop, intercept, correlated coefficient, root mean square error (RMSE), total deviation index (TDI), and the proportion of estimated GFR (eGFR) within 10% and 30% deviation of rGFR (P₁₀ and P₃₀). Of the three modified equations, the modified CKD-EPI equation showed the best accuracy.

Conclusions

Mathematical algorithms could be a considerable tool to modify the GFR equations. Accuracy of all the three modified equations was significantly improved in which the modified CKD-EPI equation could be the optimal one.     

Separation of lactic acid from acetic acid using a four-zone SMB

A simulated moving bed (SMB) process has been developed to separate l-(+)-lactic acid from acetic acid, a major impurity in the fermentation broth of Lactobacillus rhamnosus. Poly(4-vinylpyridine) resin (PVP) was selected as the adsorbent. Adsorption isotherms and mass transfer parameters of the organic acids were estimated from single-column frontal tests. Experimental results show that the Langmuir isotherms obtained from the frontal tests can be used in the design of an SMB process to achieve 99.9% purity and over 93% yield of lactic acid. The column profiles and effluent histories, however, deviate from rate model predictions based on the Langmuir isotherms. They agree more closely with the predictions based on a modified Langmuir isotherm for lactic acid. The standing wave design method for systems with modified Langmuir isotherms is developed in this study. Rate model simulations show that the process based on the modified design method can achieve high purity (>99.9%) and high yield (>99.9%). For this nonlinear system, accurate isotherm model and model parameters are needed in the design, and the zone flow rates must be closely monitored and controlled in order to ensure high purity and high yield in the SMB process.     

Effect of solid-phase chemical modification on the features of the lipase from Thermomyces lanuginosus

The lipase from Thermomyces lanuginosus (TLL) was immobilized on octyl Sepharose and further modified with ethylenediamine (EDA) after activation of the carboxylic groups with carbodiimide. Different degrees of modification of the carboxyl groups were carried out by controlling the concentration of carbodiimide (10%, 50% or 100%). Subsequently, the effect of incubation of the modified preparations on hydroxylamine to recover the modified tyrosine was also studied. The modified enzymes exhibited a mobility in native electrophoresis quite different from that of the unmodified lipase (as expected by the changes in charge), and required higher concentrations of cationic detergent to become desorbed from the support. Interestingly, the chemical modification of the immobilized TLL produced an improvement in its activity, proportional to the amination degree. This increase in activity was much more significant at pH 10, where the fully modified preparation increased the activity by a factor of 10 as compared to the unmodified preparation. Moreover, the incubation of the chemically aminated preparations in a hydroxylamine solution improved the activity by an additional factor of 1.2. The fully aminated and incubated in hydroxylamine preparation exhibited a thermostability higher than that of the unmodified preparation, mainly at pH 5 (almost a 30 fold factor). In the presence of tetrahydrofurane, some stabilization was observed at pH 7, while at pH 9 the stability of the modified enzyme decreased (under all the assayed amination degrees) when compared to that of the unmodified enzyme. Thus, this simple protocol may be a rapid and efficient way of preparing a TLL biocatalyst with higher activity and stability, although this will depend on the inactivation conditions.     

Modification of cellulase and its application to extraction of diosgenin from Dioscorea zingiberensis C.H. Wright

In order to enhance the thermostability and efficiency of cellulase in the extraction of diosgenin from Dioscorea zingiberensis C.H. Wright, we applied polyethylene glycol (PEG) (400, 1000, 2000, and 4000) to modify cellulase. The modified cellulase, α-amylase and β-glycosidase were used to hydrolyze the material. The results show that the thermostability of modified cellulase is better than that of natural cellulase, the optimum pH value and temperature of modified cellulase are wider than that of natural cellulase, the activity of cellulase modified by activated PEG2000 is higher than that of cellulase modified by other modifiers, and its remaining activity is 58% of its initial value. With this technique, the purity of the product reaches 96%, the melting point is 201–204 °C, the yield rate and the extraction rate of the diosgenin reaches 2.80% and 96.6%, respectively. IR spectra and ¹H NMR spectroscopy were used to confirm the structure of the product.     

Nonenzymatic incorporation of glucose and galactose into brain cytoskeletal proteins in vitro.

Initial studies demonstrated the loss of lysine and simultaneous appearance of glucitollysine in intracellular proteins following incubation with sugar. For example, when a crude nervous tissue cytoskeletal preparation was incubated in 100 mM glucose for 10 days, > 60% of the lysine residues were modified. Over 20% of the lysyl residues in a spinal cord neurofilament preparation are susceptible to Schiff base formation after one day and over 30% following five days of incubation with 100 mM glucose. When incubated with 100 mM galactose, F- and G-actin were found to be significantly modified in as few as 15 h, with > 70% of the lysyl residues lost. After 45 h of incubation, > 90% of the residues had been modified. These data also indicate that many of the lysyl residues in F- and G-actin are exposed and very susceptible to modification by sugar. This rapid and extensive modification of lysine in actin in vitro suggest that it may be modified in diabetic nervous tissue.     

Biochemical aspects of the visual process XXVIII. Classification of sulfhydryl groups in rhodopsin and other photoreceptor membrane proteins

Reaction of isolated bovine rod outer segment membrane with radioactiveN-ethylmaleimide, both in the presence and absence of 1% dodecyl sulfate followed by dodecyl sulfate-polyacrylamide gel electrophoresis, shows that six sulfhydryl groups (96% of total sulfhydryl in this membrane) are located on the rhodopsin molecule.On the basis of their reactivity towardsp-chloromercuribenzoate andp-chloromercuribenzene sulfonate in suspensions of outer segment membranes, the sulfhydryl groups of rhodopsin can be divided into three pairs. One pair is rapidly modified, both in light and darkness. This modification does not impair the recombination capacity of opsin with 11-cis retinaldehyde under regeneration of rhodopsin. A second pair is modified upon prolonged interaction with thep-chloromercuriderivatives in darkness. Modification of this pair leaves the typical rhodopsin absorbance at 500 nm intact, but a proportional loss of recombination capacity does occur. The third pair is only modified after illumination and is probably located in the vicinity of the chromophoric center.The difference between these results and those obtained by modification with dithiobis-(2-nitrobenzoic acid) orN-ethylmaleimide in suspension, where even upon prolonged exposure to light as well as in darkness only two sulfhydryl groups of rhodopsin are modified, is explained by the detergent-like character of thep-chloromercuri-derivatives.     

A modified model for estimating soil redistribution on grassland by using 7Be measurements

Background and aims

⁷Be has been used as a powerful tracer for estimating short-term soil redistribution by virtue of its short half-life. However, the existing conversion model associated with this radionuclide means that it can only be applied to bare soils because vegetation will intercept a large proportion of ⁷Be fallout. A modified model which takes into consideration the impact factor of vegetation was reported in this paper and the estimation of soil redistribution was compared by using both the conventional and the modified models.

Methods

Field experiment on ⁷Be distribution in above-ground grasses and soils was carried out on a 100 m² grass-covered slope. The vegetation interception factor (P) was determined and the soil redistribution rates were calculated by using the previous model and the modified model.

Results

The result shows that nearly 40 % of the atmospherically deposited ⁷Be will be sequestered by leaf surfaces of herbaceous plants. Soil loss rates on grassland will be remarkably overestimated by using the previous model.

Conclusions

The net soil loss estimated from the modified model is more accurate than that derived from the conventional model and the modified model will be more appropriate to estimate soil redistribution rates on soils with significant vegetation cover by using ⁷Be technique.     

A comparative study of blood culture conventional method vs. a modified lysis/centrifugation technique for the diagnosis of fungemias

The purpose of this work was to compare the efficacy of blood culture conventional method vs. a modified lysis/centrifugation technique. Out of 450 blood specimens received in one year, 100 where chosen for this comparative study: 60 from patients with AIDS, 15 from leukemic patients, ten from febrile neutropenic patients, five from patients with respiratory infections, five from diabetics and five from septicemic patients. The specimens were processed, simultaneously, according to the above mentioned methodologies with daily inspections searching for fungal growth in order to obtain the final identification of the causative agent. The number (40) of isolates recovered was the same using both methods, which included; 18 Candida albicans (45%), ten Candida spp. (25%), ten Histoplasma capsulatum (25%), and two Cryptococcus neoformans (5%). When the fungal growth time was compared by both methods, growth was more rapid when using the modified lysis/centrifugation technique than when using the conventional method. Statistical analysis revealed a significant difference (p<0.05) between them. The modified lysis/centrifugation technique showed to be more efficacious than the conventional one, and therefore the implementation of this methodology is highly recommended for the isolation of fungi from blood.     

Purification of Leucine tRNA Isoaccepting Species from Soybean Cotyledons: I. Benzoylated Diethylamino Cellulose Fractionation, N-Hydroxysuccinimide Modification, and Characterization of Product

Transfer RNA from soybean (Glycine max) cotyledons was purified to homogeneity followed by the purification of the family of leucine tRNA via benzoylated diethylaminoethyl cellulose (BDC) chromatography. Nonacylated total purified tRNA was salicylhydroxamate (SHAM) modified by the phenoxyacetyl method and fractionated into three peaks on a BDC column. The first peak containing bulk tRNA with no hydrophobic character amounted to 78% of the added tRNA. The second peak containing 19% of the added tRNA and represents the tRNA with intrinsic hydrophobic properties. The third peak containing 3% of the tRNA represents the SHAM modified tRNA and nonspecifically modified tRNA. Transfer RNA peaks I and II were pooled and subsequently stoichiometrically acylated in two batches, one containing [¹⁴C]leucine while the other contained unlabeled leucine. The acylated tRNA was loaded on and step-eluted from a BDC column. The purified acylated-tRNA was phenoxyacetyl modified and following ethanol precipitation was fractionated on a BDC column. A double peak eluted from the column in the ethanol gradient contained 5.3% of the starting optical density and 85.3% of the starting counts per minute. Characterization of this leucine tRNA showed typical ultraviolet spectra properties and appeared to be homogeneous on a G-100 Sephadex column. The minimum purity of the tRNA was 32 to 35%. Finally, the acylated tRNA was chromatographed on an RPC-2 column giving six leucine isoaccepting tRNAs. The data indicate that leucine tRNA was highly purified without losing the integrity of the family of isoacceptors.     

Efficacy of immunohistochemical staining in detecting Helicobacter pylori in Saudi patients with minimal and atypical infection

Gastric Helicobacter pylori infection is diagnosed based on histopathological evaluation of gastric mucosal biopsies, urease test, urea breath test, H. pylori culturing, or direct detection using polymerase chain reaction (PCR). This study aimed to evaluate the efficacy of immunohistochemical (IHC) staining in detecting H. pylori in gastric biopsies from patients with chronic gastritis and minimal or atypical infection. Gastric biopsies from 50 patients with chronic gastritis were subjected to routine haematoxylin and eosin (H&E), modified Giemsa, and IHC staining. The results of staining were compared with those of quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis identified 32 (64%) H. pylori-positive cases, whereas IHC, H&E, and modified Giemsa staining identified 29 (58%), 27 (54%), and 21 (42%) positive cases. The sensitivity of IHC staining (87.50%) was higher than that of H&E (59.38%) and modified Giemsa (43.75%) staining. The specificity of H&E, modified Giemsa, and IHC staining was 55.56%, 61.11%, and 94.44%, respectively. IHC staining exhibited the highest diagnostic accuracy (90%), followed by H&E (58%) and modified Giemsa (50%) staining. Active gastritis, intestinal metaplasia, and lymphoid follicles were detected in 32 (64%), 4 (8%), and 22 (44%) cases, respectively, and all of these cases were H. pylori positive. In contrast to routine H&E and modified Giemsa staining, IHC allows for the accurate H. pylori detection in cases with minimal or atypical infection. Moreover, IHC can be an alternative diagnostic method to qRT-PCR for detection of H. pylori in such cases.Key words: Helicobacter pylori, immunohistochemistry, modified Giemsa stain, real-time polymerase chain reaction, chronic gastritis     

Efficiency of six different octenol-baited traps for collecting horseflies (Diptera: Tabanidae)

The efficiency and the potential species preference for six different horsefly trap types was studied. A total of 2401 horseflies were collected during this study. The most effective trap was the modified box trap with (27.9%), followed by the Nzi trap with (24.5%), Malaise trap (Townes design 1972) with (22.5)%, canopy trap with (17.9%), modified small canopy trap with (7.0%) and Malaise trap (Townes design 1962) with (0.2%). The Nzi and Malaise trap (Townes design 1972) did not differ significantly in the number of collected horseflies. The comparison of all other traps revealed significant differences in the number of collected horseflies. Tabanus bromius was the most abundant species with (58.6%) of the collected sample. The modified box trap was the most successful in collecting of T. bromius, while the Malaise trap (Townes design 1972) was the most successful in collecting the species Haematopota pluvialis, T. maculicornis and T. tergestinus. This study demonstrated that different trap designs influenced the quantity, species and taxonomic diversity of horseflies that were collected. Variables in the trap design included cloth type and colour and size and shape of trap. More detailed studies should be done to determine which factor or factors are most significant.     

Optimization and scale-up of a new photobleaching agar extraction process from Gracilaria lemaneiformis

An eco-friendly photobleaching extraction process for agar extraction from the red alga Gracilaria lemaneiformis was developed for the benefit of workers’ health and environmental safety. Here we report the optimization of key process parameters (alkali modification concentration, photobleaching duration, algal length and screen filter opening size) in order to scale up this new technique. The optimal conditions were found to be modification by 3–5% NaOH, photobleaching for 5 h, using algal fragments 2 –4 cm in length, and a filter screen with a 6 μm opening. A 20-L agar extraction reactor was thus constructed, and the scale-up of the agar extraction process was tested in six batch experiments. The resulting agar quality was similar to that of the laboratory-scale extraction. In addition, batch-to-batch reproducibility was excellent. The results demonstrate the excellent scale-up ability and potential application of this new photobleaching agar extraction process on a commercial scale. The agar yield and gel strength for 5% NaOH modified agar were 26.8% and 1,897 g cm⁻², while those for 3% NaOH modified agar were 28.2% and 1,287 g cm⁻², respectively. It is clear that the agar yield and quality can be manipulated via alkali modification in this new eco-friendly extraction to meet market demands.     

Chemical modification of arginine residues in alpha-bungarotoxin.

The reaction of alpha-bungarotoxin (alpha-BuTX) with 1,2-cyclohexanedione resulted in the modification of only Arg-72 but arginine at position 36 or 72, as well as both were modified by reaction of the toxin with p-hydroxyphenylglyoxal. No derivative modified at Arg-25 was obtained, indicating that this residue may be located in the interior region of alpha-BuTX molecule. Monoderivative at Arg-72 showed about 50% of the lethal toxicity and binding activity of alpha-BuTX to nicotinic acetylcholine receptor (AChR), while the activity was decreased to one-third when the invariant Arg-36 was modified, indicating that the latter residue is more closely related to the interaction of the toxin with AChR. Approx. 13% of the residual activity was observed when both arginine residues at 36 and 72 were modified. The antigenicity of alpha-BuTX was still retained essentially intact after Arg-36 or -72 was modified, whereas it decreased to 50% when both these arginine residues were modified. The present study indicates that Arg-36 and -72 in alpha-BuTX may be involved in the multipoint contact between the toxin and AChR, but neither is absolutely essential for the binding.     

Modification of corn cob meal with quarternary ammonium groups

Corn cob meal was modified with quarternary ammonium groups and subsequently extracted with 80% ethanol, water, and 5% NaOH. The fractions obtained had lower polydispersities, values, and yields than unmodified material. The yields are lower than those obtained on bagasse under the same conditions. The modification caused the drastic degradation of the ethanol-lignin (EL) fraction. The one-step extraction with NaOH/H₂O₂ gave 28·8% yield of material (calculated on the starting material) which contained 12·0% Klason lignin, and had the highest polydispersity (4·3, ). The water-soluble fractions consisted of arabinoglucuronoxylan and alkali-soluble fractions of xylan without other sugar moieties. The water-soluble fraction from NaOH/H₂O₂ extraction contained arabinoglucuronoxylan with modified arabinose and acid units. By this method higher yields could be obtained than on bagasse treated by the sequential extraction.     

Synergism of natural and constructed wetlands in Beijing,China

An integrated wetland system (IWS) including constructed wetlands (CWs) and modified natural wetlands (NWs) for wastewater treatment to replenish water to wetlands located at the Beijing Wetland School (BWS) in Beijing, China, is presented in this paper. The synergistic effects of CWs and NWs on treated water quality are investigated. The IWS is proved to be an effective wastewater treatment technique and a better alternative to alleviate the water shortage for conservation of wetlands based on the monitoring data obtained from October 2007 to 2008. The results show that CWs and NWs play different roles in removing contaminants from wastewater. The COD removal efficiency in CWs is higher than that in modified NWs, whereas the modified NWs can compensate for the deficiency of CWs where a stable and sufficient rhizosphere is not fully formed in the start-up period. All removal rates of COD, TN, and TP in CWs and modified NWs vary from 50 to 70%, while the total removal rate of COD, TN, and TP in IWS is about 85–90%. The operational results show that the maximum area loading of organic pollutants in modified NWs (65 kg/ha d) is slightly higher than the empirical one (60 kg/ha d) recommended by USEPA (2000) for free water surface wetlands.     

Modified Polysaccharides as Fast Disintegrating Excipients for Orodispersible Tablets of Roxithromycin

The purpose of this study was to develop a dosage form that was easy to administer and provides rapid release of the drug roxithromycin, using modified polysaccharides as rapidly disintegrating excipients. Modified polysaccharides co grinded treated agar (C-TAG) and co grinded treated guar gum (C-TGG) were prepared by subjecting pure polysaccharides namely agar and guar gum respectively to sequential processes of wetting, drying and co grinding with mannitol (1:1). The modified polysaccharides were characterized by Scanning Electron Microscopy and Diffuse Reflectance Spectroscopy and evaluated for particle size distribution, derived properties, swelling index and biodegradability. Optimization studies based on 2² factorial designs, with friability and disintegration time as response parameters were used to formulate orodispersible tablets of roxithromycin and evaluated for wetting time, water absorption ratio and in vitro drug release at salivary pH 6.4 and physiological pH 7.4. Results indicated that lower levels of modified polysaccharides namely C-TAG in F₃ and C-TGG in F₇ and higher levels of microcrystalline cellulose, exhibited least disintegration times without friability concerns. In vitro release of optimized formulations F₃ and F_7, both at salivary pH and physiological pH was found to be more than 90% within 30 min as compared to 27.82% at the same time point of conventional formulation. Stability studies carried out as per ICH Q1A guidelines suggested the formulations to be stable for a period of 6 months. Thus the approach of using modified polysaccharides as fast disintegrating excipient can be used to formulate a stable orodispersible formulation.