Iron porphyrin-based porous organic polymer with high peroxidase-like activity as colorimetric sensor for glutathione and ascorbic acid assay.

A new iron porphyrin-based organic polymer (Fe-POP) was synthesized through the William ether reaction. The as-prepared Fe-POP presented high chemical stability, wide pore distribution, high iron content, and strong affinity with 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2), which contributed to its excellent peroxidase-mimicking performance. In the presence of H2O2, Fe-POP could catalyze the transparent TMB into blue ox-TMB, which could be easily distinguished by the naked eyes. Moreover, glutathione (GSH) and ascorbic acid (AA) could convert blue ox-TMB into colorless TMB due to the inhibitory effect of GSH/AA to the catalytic oxidation of TMB. Based on this phenomenon, a rapid and sensitive colorimetric method for the assay of H2O2, GSH, and AA was developed using Fe-POP as sensor. The detection limits of H2O2, GSH, and AA  were 1.37, 0.44, and 0.33 µM, respectively. Finally, the colorimetric method based on Fe-POP was used to evaluate the GSH and AA content in real samples, which provided the guidance for GSH and AA supplements in our daily diet, suggesting the significant potential of Fe-POP in practical applications.

Magnetically controlled colorimetric aptasensor for chlorpyrifos based on copper-based metal-organic framework nanoparticles with peroxidase mimetic property.

The fabrication of a magnetically controlled colorimetric aptasensor for chlorpyrifos is reported. The aptasensor was fabricated by the attachment of the colorimetric labels onto the magnetic carrier due to the hybridization reaction between the complementary DNA and aptamer. Chlorpyrifos detection was realized by monitoring the color changes of the TMB/H2O2 solution before and after incubation of the aptasensor with chlorpyrifos via exposure to external magnetic force. The color change was monitored at 650 nm by UV-Vis spectrophotometer. Under the optimal conditions, this magnetically controlled Cu-MOF-based aptasensor showed a detection limit of 4.4 ng/mL with a linear range of 0-1250 ng/mL. The colorimetric aptasensor displayed high selectivity for chlorpyrifos toward other interfering pesticides. The aptasensor was successfully applied for the spiked test of chlorpyrifos in fruits and vegetable samples with good recovery, which were in agreement with data obtained by GC-MS analysis. This magnetically controlled Cu-MOF-based sensing strategy not only leads to development of efficient and facile phase separation, but also expands the MOF's target scope from H2O2 or glucose to pesticides. Graphical abstract.

A nanozyme-based cascade colorimetric aptasensor for amplified detection of ochratoxin A.

Colorimetric assays have been widely developed for the detection of toxin ochratoxin A (OTA), but most of them suffer from moderate sensitivity when they are adopted for the detection of trace OTA in a complicated food matrix. For the purpose of overcoming this issue, an innovative cascade reaction-based colorimetric aptasensor was developed for the achievement of high sensitivity. The biotin-labelled OTA aptamer was immobilized onto streptavidin magnetic beads by means of the biotin-streptavidin reaction. With OTA binding to its aptamer, the structural switching of the aptamer results in the release of the alkaline phosphatase-labelled oligonucleotide, which is partially complementary to the aptamer. Following the magnetic separation, the cascade reaction is initiated through the enzymatic conversion of ascorbic acid-2-phosphate into ascorbic acid. Subsequent to that, the generated ascorbic acid reduces MnO2 nanosheets to Mn2+ ions, accordingly destroying the oxidase-mimicking activity of MnO2 nanosheets. In consequence, it is not possible to oxidize 3,3',5,5'-tetramethylbenzidine (TMB), a substrate for oxidase, with Mn2+ for the production of the blue colour product (TMB Ox). With the increasing amount of OTA, a colour change occurs from blue to colourless. The cascade reaction has the potential of greatly amplifying the detection signal, together with remarkably improving the sensitivity, making this colorimetric sensor a universal and promising platform for the highly sensitive detection of mycotoxins in the field of public food safety monitoring.

Aptamer-Structure Switch Coupled with Horseradish Peroxidase Labeling on a Microplate for the Sensitive Detection of Small Molecules.

Detection of small molecules with good sensitivity, high throughput, simplicity, and generality using aptamers is desired but still remains challenging. We described an aptamer-structure-switch assay coupled with horseradish peroxidase (HRP) labeling on microplates for sensitive absorbance and chemiluminescence detection of small molecules. This assay relies on competition for affinity binding to a limited HRP-labeled aptamer between small-molecule targets and immobilized short DNA strands complementary to the aptamer (cDNA) on a microplate. In the absence of targets, the HRP-labeled aptamer hybridizes with the cDNA on the microplate, and HRP catalyzes substrate into product, generating absorbance or chemiluminescence signals. The binding of small-molecule targets to aptamers causes displacement of HRP-labeled aptamers from the cDNA and signal decrease. In chemiluminescence-analysis mode, the assay achieved detection of aflatoxin B1 (AFB1), ochratoxin A (OTA), and adenosine triphosphate (ATP) with detection limits of 10 pM, 20 pM, and 20 nM, respectively. This assay does not require enzyme-labeled small molecules or the conjugation of small molecules on solid phase. HRP, as an enzyme label, here allows for easily obtainable and highly active signal amplification. This microplate assay is rapid and promising for high-throughput analysis. It shows potential for wide applications in the detection of small molecules.

Colorimetric and electrochemical (dual) thrombin assay based on the use of a platinum nanoparticle modified metal-organic framework (type Fe-MIL-88) acting as a peroxidase mimic.

An electrochemical and colorimetric dual-readout method is described for the determination of thrombin. A platinum nanoparticle (Pt NP) modified metal organic framework (MOF) acts as a peroxidase (POx) mimic that causes the formation of a blue product from 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide, with an absorption maximum at 650 nm. In addition, gold nanoparticles enrich initiators that trigger the hybridization chain reaction for dual signal amplification to generate an electrochemical current typically measured at 0.31 V (from -0.5 to -0.1 V) and allow quantitation of thrombin with high sensitivity and over a wide detection range. The colorimetric and electrochemical (dual) thrombin assay produces two kinds of signals which warrants accuracy, diversity, and an option for visual inspection. The dual-channel sensor allows for the quantitative determination of thrombin with a low detection limit (0.33 fM) for the electrochemical method and 0.17 pM for the colorimetric method) and over a wide detection range (1 fM to 10 nM for electrochemical method and 0.5 pM to 1 nM for colorimetric method). The electrochemical detection limit is lower than that of colorimetry, and the linear range is wider, which is more suitable for further quantitative analysis of the target. Graphical abstract Schematic representation of a colorimetric and electrochemical (dual) thrombin assay based on the use of a platinum nanoparticle modified metal-organic framework for color development and hybridization chain reaction for electrochemical signal. C-TBA: complementary sequences of thrombin aptamer, TBA: thrombin aptamer, I-Au NPs: initiators enriched by gold nanoparticles, S-AuE: sensing gold electrode, RS-AuE: reacted sensing gold electrode, TB: thrombin, MB: Methylene Blue, HCR: hybridization chain reaction.

A colorimetric assay for acetylcholinesterase activity and inhibitor screening based on the thiocholine-induced inhibition of the oxidative power of MnO2 nanosheets on 3,3',5,5'-tetramethylbenzidine.

The authors describe a colorimetric method for the determination of the activity of acetylcholinesterase (AChE). Manganese dioxide (MnO2) nanosheets directly reacts with 3,3',5,5'-tetramethylbenzidine (TMB) in the absence of hydrogen peroxide (H2O2). This leads to the formation of a blue product (oxTMB) with an absorption peak at 652 nm. If AChE hydrolyzes its substrate acetylthiocholine chloride, thiocholine is formed which blocks the oxidative power of the MnO2 nanosheets. Hence, oxTMB will not be formed. The decreased absorbance is directly related to the AChE activity in the 0.01-1.0 mU·mL-1 range. The detection limit is 0.01 mU·mL-1 and the relative standard deviation is 1.2% (for n = 11 at 0.5 mU·mL-1). The method was also applied to screen for inhibitors of AChE. Graphical abstract Based on the oxidizing properties of manganese dioxide nanosheets (MnO2 nanosheets), we report a colorimetric method for determining acetylcholinesterase activity with the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB).

A conventional chemical reaction for use in an unconventional assay: A colorimetric immunoassay for aflatoxin B1 by using enzyme-responsive just-in-time generation of a MnO2 based nanocatalyst.

The authors describe a colorimetric immunoassay for the model nalyte aflatoxin B1 (AFB1). It is based on the just-in-time generation of an MnO2 nanocatalyst. Unlike previously developed immunoassay, the chromogenic reaction relies on the just-in-time formation of an oxidase mimic without the aid of the substrate. Potassium permanganate (KMnO4) is converted into manganese dioxide (MnO2) which acts as an oxidase mimic that catalyzes the oxidation 3,3',5,5'-tetramethylbenzidine (TMB) by oxygen to give a blue colored product. In the presence of ascorbic acid (AA), KMnO4 is reduced to Mn(II) ions. This results in a decrease in the amount of MnO2 nanocatalyst. Hence, the oxidation of TMB does not take place. By adding ascorbate oxidase, AA is converted into dehydroascorbic acid which cannot reduce KMnO4. Based on these observations, a colorimetric competitive enzyme immunoassay was developed where ascorbate oxidase and gold nanoparticle-labeled antibody against AFB1 and magnetic beads carrying bovine serum albumin conjugated to AFB1 are used for the determination of AFB1. In presence of AFB1, it will compete with the BSA-conjugated AFB1 (on the magnetic beads) for the labeled antibody against AFB1 on the gold nanoparticles. This makes the amount of ascorbate oxidase/anti-AFB1 antibody-labeled gold nanoparticles, which conjugated on magnetic beads, reduce, and resulted in an increase of ascorbic acid. Under optimal conditions, the absorbance (measured at 652 nm) decreases with increasing AFB1 concentrations in the range from 0.1 to 100 ng mL-1, with a 0.1 ng mL-1 detection limit (at the 3Sblank level). The accuracy of the assay was validated by analyzing spiked peanut samples. The results matched well with those obtained with a commercial ELISA kit. Conceivably, the method is not limited to aflatoxins but has a wide scope in that it may be applied to many other analytes for which respective antibodies are available. Graphical abstract Schematic illustration of ascorbate oxidase (AOx)-mediated potassium permanganate (KMnO4)-responsive ascorbic acid (AA) for visual colorimetric immunoassay of aflatoxin B1 (AFB1) by coupling with hydrolytic reaction of AOx toward AA and the KMnO4-Mn(II)-TMB system [note: 3,3',5,5'-tetramethylbenzidine: TMB].

A simple and novel colorimetric assay for tyrosinase and inhibitor screening using 3,3',5,5'-tetramethylbenzidine as a chromogenic probe.

A novel colorimetric method for the detection of tyrosinase (TYR) and its inhibitor by taking utilization of Ag+-3,3',5,5'-tetramethylbenzidine (TMB) detection system has been proposed. Ag+ could oxidize TMB to oxidized TMB (oxTMB) and induce a blue color solution corresponding to an absorption peak centered at 652nm. The addition of dopamine (DA) could cause the reduction of oxTMB which resulted in the fading of the blue color and a decrease of the absorbance at 652nm. However, in the presence of TYR, DA could be oxidized to dopaquinone, which inhibited the reduction of oxTMB by DA, resulting in a blue color recovery and an increase of the absorbance at 652nm. Based on this finding, we propose a method to quantitatively detect TYR activity with the help of UV-vis spectroscopy. The developed assay is highly sensitive with a low detection limit of 0.010U/mL. More importantly, this method is fairly simple and inexpensive without the use of complicated nanomaterials. In addition, it constructs a useful platform for TYR inhibitor screening.

Proteomic Analysis Reveals the Dynamic Role of Silicon in Alleviation of Hyperhydricity in Carnation Grown In Vitro.

The present study depicted the role of silicon in limiting the hyperhydricity in shoot cultures of carnation through proteomic analysis. Four-week-old healthy shoot cultures of carnation "Purple Beauty" were sub-cultured on Murashige and Skoog medium followed with four treatments, viz. control (-Si/-Hyperhydricity), hyperhydric with no silicon treatment (-Si/+Hyperhydricity), hyperhydric with silicon treatment (+Si/+Hyperhydricity), and only silicon treated with no hyperhydricity (+Si/-Hyperhydricity). Comparing to control morphological features of hyperhydric carnations showed significantly fragile, bushy and lustrous leaf nature, while Si supply restored these effects. Proteomic investigation revealed that approximately seventy protein spots were differentially expressed under Si and/or hyperhydric treatments and were either up- or downregulated in abundance depending on their functions. Most of the identified protein spots were related to stress responses, photosynthesis, and signal transduction. Proteomic results were further confirmed through immunoblots by selecting specific proteins such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), PsaA, and PsbA. Moreover, protein-protein interaction was also performed on differentially expressed protein spots using specific bioinformatic tools. In addition, stress markers were analyzed by histochemical localization of hydrogen peroxide (H2O2) and singlet oxygen (O21-). In addition, the ultrastructure of chloroplasts in hyperhydric leaves significantly resulted in inefficiency of thylakoid lamella with the loss of grana but were recovered in silicon supplemented leaves. The proteomic study together with physiological analysis indicated that Si has a substantial role in upholding the hyperhydricity in in vitro grown carnation shoot cultures.

A triple-amplification colorimetric assay for antibiotics based on magnetic aptamer-enzyme co-immobilized platinum nanoprobes and exonuclease-assisted target recycling.

Herein, an ultrasensitive and selective colorimetric assay for antibiotics, using chloramphenicol (CAP) as the model analyte, was developed based on magnetic aptamer-HRP-platinum composite probes and exonuclease-assisted target recycling. The composite probes were prepared through immunoreactions between the double stranded DNA antibody (anti-DNA) labeled on core-shell Fe3O4@Au nanoparticles (AuMNP-anti-DNA) as the capture probe, and the double stranded aptamer (aptamer hybrid with its complementary oligonucleotides) labeled on Pt@HRP nanoparticles as the nanotracer (ds-Apt-HRP-PtNPs). When the CAP samples were incubated with the probes for 30 min at room temperature, they could be captured by the aptamer to form a nanotracer-CAP complex, which was then released into the supernatant after magnetic separation. This is because the anti-DNA on the capture probes cannot recognize the single strand aptamer-CAP complex. The exonuclease I (Exo I) added into the supernatant can further digest the aptamer-CAP from the 3'-end of the aptamer and the CAP in the aptamer-CAP complex can be released again, which can further participate in a new cycling process to react with the probes. Pt and HRP in the nanotracer could both catalyze and dual amplify the absorbance at 650 nm ascribed to the 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 system. Moreover, Exo I can assist the target recycling, which can further amplify the signal. Thus, the triple amplified signal can be quantified by ultraviolet-visible spectroscopy. The experimental results showed that the CAP detection possessed a linear range of 0.001-10 ng mL(-1) and a detection limit of 0.0003 ng mL(-1) (S/N = 3). The assay was successfully employed to detect CAP in milk, which is much more facile, time saving, and sensitive than the commercial ELISA kits.

Double enzymatic cascade reactions within FeSe-Pt@SiO2 nanospheres: synthesis and application toward colorimetric biosensing of H2O2 and glucose.

A facile process was developed for the synthesis of FeSe-Pt@SiO2 nanospheres based on the hydrothermal treatment of FeCl3·6H2O, selenium and NaBH4 in ethanolamine solvent, followed by reducing HPtCl4 with NaBH4 in the presence of FeSe particles to obtain FeSe coated with Pt NPs (FeSe-Pt), ending with a surfactant assembled sol-gel process to obtain FeSe-Pt@SiO2. The morphology and composition of FeSe-Pt@SiO2 were characterized by transmission electron microscopy, high resolution TEM, X-ray diffraction and Fourier transform infrared spectroscopy. Structural analyses revealed that FeSe-Pt@SiO2 nanospheres were of regular spherical shape with smooth surfaces due to the SiO2 shells, compared with FeSe particles with 150 nm lateral diameter. The prepared FeSe-Pt@SiO2 nanospheres possessed both intrinsic glucose oxidase (GOx-) and peroxidase-mimic activities, and we engineered an artificial enzymatic cascade system with high activity and stability based on this nanostructure. The good catalytic performance of the composites could be attributed to the synergy between the functions of FeSe particles and Pt NPs. Significantly, the FeSe-Pt@SiO2 nanospheres as robust nanoreactors can catalyze a self-organized cascade reaction, which includes oxidation of glucose by oxygen to yield gluconic acid and H2O2, and then oxidation of 3,3,5,5-tetramethylbenzidine (TMB) by H2O2 to produce a colour change. Colorimetric detection of H2O2 and glucose using the FeSe-Pt@SiO2 nanospheres was conducted with high detection sensitivities, 0.227 nM and 1.136 nM, respectively, demonstrating the feasibility of practical sensing applications. It is therefore believed that our findings in this study could open up the possibility of utilizing FeSe-Pt@SiO2 nanospheres as enzymatic mimics in diagnostic and biotechnology fields.

Proteomic identification of a basic peroxidase stabilized within acetylated polymannan polysaccharide of Aloe barbadensis.

Acetylated polymannan polysaccharide (ApmP) isolated from Aloe barbadensis Miller contains a stable peroxidase that was solubilized to investigate its biochemical, electrophoretic, immunological, and proteomic properties. In the electrophoretic band corresponding to the solubilized peroxidase, proteomic analysis detected seven tryptic peptides that matched homologous peptides covering one third of the ATP22a peroxidase of Arabidopsis thaliana. All the characteristics tested indicated that the activity stabilized within the ApmP pertains to the basic secretory peroxidase family, which includes members that have several biotechnological uses. Hence ApmP might yield a widely used peroxidase in stabilized form.

Occupational exposure to chemical and petrochemical industries and bladder cancer risk in four western Canadian provinces.

Occupational factors have been proposed to play a critical role in bladder cancer. This population-based case-control study was conducted to confirm the association between selected occupational and non-occupational risk factors and risk of bladder cancer using data collected from the four western Canadian provinces. Unconditional logistic regression analyses were based on 549 histologically confirmed bladder cancer cases and 1099 controls. Bladder cancer risk was found to increase with increasing pack-years of cigarette smoking with an odds ratio (OR) in the highest quartile of 3.32 (95% confidence interval [CI], 2.28-4.82). A dose-response relationship was demonstrated between bladder cancer and pack-years of smoking (p < 0.0001). A positive trend was observed with coffee consumption in men (p < 0.0001), with the highest risk in the highest category of exposure: drinkers of four cups or more per day had an OR of 1.77 (95% CI 1.11-2.82). Increased bladder cancer risk was associated with self-reported exposure at work to several chemicals: asbestos (OR 1.69 [95% CI 1.07-2.65]); mineral, cutting or lubricating oil (1.64 [95% CI 1.06-2.55]); benzidine (2.20 [95% CI 1.00-4.87]). The population attributable fraction (PAF) estimates were 51% for cigarette smoking, 17% for heavy coffee consumption, 10% for mineral, cutting or lubricating oil exposure, 6% for asbestos exposure, and 1% for benzidine exposure. Although self-reported chemical exposures have important limitations, the findings are suggestive of increased risk for several associations previously reported between chemical agents or industries and risk of bladder cancer.

Mutant spectra analysis at hisG46 in Salmonella typhimurium strain YG1029 induced by mammalian S9- and plant-activated aromatic amines.

Mutant spectra analysis was conducted with spontaneous hisG46 revertants of Salmonella typhimurium strain YG1029 and revertants induced by the plant- and mammalian S9-activation of benzidine and 4-aminobiphenyl (4-ABP). Under preincubation conditions, YG1029 cells were exposed to benizidine or 4-ABP with mammalian S9 activation or to a high molecular weight fraction that contained the plant-activated products. The induced revertants were isolated at mutagen concentrations that caused an increased mutant frequency of approximately 4- to 10-fold above background. Genomic DNA from each revertant was isolated and the hisG region was amplified using polymerase chain reaction (PCR). Using a series of specific probes and a modified version of the ECL3's-oligolabelling and detection system, each of the six possible base-pair substitution mutations at hisG46 that leads to a reversion event was determined. Of the YG1029 spontaneous revertants, transition mutations were 31.8% and transversion mutations were 68.2%. The YG1029 spontaneous mutant spectrum differed significantly from the spontaneous spectrum of TA1535 but did not significantly differ from the spontaneous TA100 mutant spectrum. The differences of the spontaneous mutant spectra among these highly related strains illustrate that the introduction of the plasmid pKM101 into S. typhimurium increased the frequency of transversions (CCC-->ACC; CCC-->CAC) and reduced site 2 (CCC-->CTC) transitions. With plant-activated benzidine, 21.1% of recovered revertants resulted from transitions and 78.9% from transversions while S9 activated-benzidine induced revertants were recovered as 14.2% from transition and 85.8% from transversion mutations. Plant-activated 4-ABP recovered 20.0% transitions and 80.0% transversions. S9-activated 4-ABP-induced 21.4% transitions and 78.6% transversions. Chi-square analysis of mutant spectra indicated that the DNA lesions that resulted in reversion at the hisG46 allele induced by plant-activated benzidine or 4-ABP were different from those generated after mammalian S9 activation of these promutagens. The plant-activated benzidine and 4-ABP induced statistically identical mutant spectra. Also, the mammalian-activated benzidine and 4-ABP induced statistically similar mutant spectra. These data show that the plant-activated and mammalian-activated aromatic amine products inflicted different types or distributions of DNA lesions that were reflected in the resulting induced mutant spectra.

Identification of catalase-like activity from Mycobacterium leprae and the relationship between catalase and isonicotinic acid hydrazide (INH).

As Mycobacterium leprae proliferate inside macrophages, it has been speculated that catalase encoded by katG may protect the bacilli from deleterious effects of peroxide generated from the macrophage and may also play a crucial role in the survival of M. leprae in vivo. However, unlike that of M. tuberculosis, the katG of M. leprae has been reported to be a pseudogene, implicating that isoniazid, which is activated to a potent tuberculocidal agent by catalase, is unlikely to be of therapeutic benefit to leprosy patients. These results raise a question as to how M. leprae avoids H202-mediated killing inside macrophages. To understand the survival of M. leprae in macrophages, the present study attempted to detect catalase-like activity in M. leprae. Catalase-like activity was found in M. leprae cell lysate by the diaminobenzidine (DAB) staining method with non-denaturing polyacrylamide gel electrophoresis. An ammonium sulphate precipitation study revealed that the catalase-like activity was precipitable with 80% ammonium sulphate. The effect of isoniazid (INH) on M. leprae growth was also tested by RT-PCR and radiorespirometric assay to examine catalase-like activity in M. leprae, because INH was activated by catalase. It was found that the viability of M. leprae was decreased at a concentration of 20 microg/ml by radiorespirometric assay and it was inhibited at higher concentrations as determined by RT-PCR. These data suggest that a catalase-like activity other than that encoded by katG is present in M. leprae.

Comparison of wheat germ agglutinin-horseradish peroxidase and biotinylated dextran for anterograde tracing of corticospinal tract following spinal cord injury.

Established methods for monitoring regeneration of the corticospinal tract involve anterograde labelling of the cortical motor neuron. While wheat germ agglutinin-horseradish peroxidase conjugate has been used to anterogradely label these neurons, we demonstrate that this technique may not completely label the whole axon and fine terminal processes when this tracer is administered in dried form. An alternative method is described for anterograde labelling of cortical motor neurons using biotinylated dextran. This tracer may be applied by either microinjection of 10% biotinylated dextran or implanting small globules of the dried tracer into the motor cortex. While more laborious, microinjection results in better anterograde labelling than implantation of dried biotinylated dextran. A procedure is also described for preparing serial coronal sections through the entire spinal cord and thaw-mounted on a minimum number of slides. The labelled nerve processes in these tissue sections can be visualised in the spinal cord under a fluorescent microscope following incubation with cy3-streptavidin complex. Permanent labelling of the biotinylated nerve processes is achieved by incubation of tissue sections with streptavidin-horseradish peroxidase conjugate followed by stringent washes and staining with tetramethylbenzidine. Use of tetramethylbenzidine allows resolution of a greater number of finer labelled processes than diaminobenzindine and allows clear visualisation of individual regenerating corticospinal tract processes. Using these procedures, we demonstrate that the corticospinal tract is completely lesioned by a standardised contusion spinal cord injury produced by the New York University weight-drop device.

Myeloperoxidase activity of the large intestine in an equine model of acute colitis.

OBJECTIVE: To determine whether quantification of myeloperoxidase (MPO) activity could be a useful laboratory technique to detect granulocyte infiltration in equine intestinal tissues. SAMPLE POPULATION: Intestinal tissue (inflamed or healthy) collected from 16 age- and sex-matched Shetland Ponies. PROCEDURE: Intestinal tissue MPO activity was determined, and histologic assessment of adjacent specimens from healthy and inflamed intestine was done. RESULTS: Intestinal tissue MPO activity and histopathologic score increased with time after castor oil challenge and peaked at 16 hours in an equine diarrhea model in which individual ponies provided their own control tissues. CONCLUSIONS: Intestinal tissue inflammation scores correlated positively with tissue MPO activity in adjacent specimens. CLINICAL RELEVANCE: Tissue MPO assay may be a useful laboratory tool to quantify intestinal mucosal inflammation in ponies.

Thalamic input to parvalbumin-immunoreactive GABAergic interneurons: organization in normal striatum and effect of neonatal decortication.

The neocortex and thalamus send dense glutaminergic projections to the neostriatum. The neocortex makes synaptic contact with spines of striatal projection neurons, and also targets a distinct class of GABAergic interneurons immunoreactive for the calcium-binding protein parvalbumin. We determined whether the parafascicular thalamic nucleus also targets striatal parvalbumin-immunoreactive interneurons. The anterograde tracer biotinylated dextranamine was injected into the parafascicular nucleus of adult rats. Double-labeled histochemistry/immunohistochemistry revealed overlapping thalamic fibers and parvalbumin-immunoreactive neurons in the neostriatum. Areas of overlap within the sensorimotor striatum were analysed by electron microscopy. Of 311 synaptic boutons originating from the parafascicular nucleus, 75.9% synapsed with unlabeled dendrites, 22.5% with unlabeled spines, and 1.3% had parvalbumin-immunoreactive dendrites as a postsynaptic target. Only 4% of all asymmetric synapses on parvalbumin-immunoreactive dendrites were derived from the parafascicular nucleus. A separate group of animals underwent bilateral neocortical deafferentation on the third postnatal day, prior to injection of anterograde tracer into the parafascicular nucleus of adult animals. These experiments were performed with the dual purpose of (i) reducing the possibility that thalamic inputs to parvalbumin-immunoreactive neurons are the result of transsynaptic uptake of tracer by a thalamo-cortico-striatal route, and (ii) determining whether competitive interactions between developing corticostriatal and thalamostriatal fibers may account for the relatively sparse thalamic input onto parvalbumin-immunoreactive interneurons. In decorticates, 219 striatal synaptic contacts derived from the parafascicular nucleus, out of which 77.2% were on unlabeled dendrites, 20.9% were upon unlabeled spines, and 0.9% targeted parvalbumin-immunoreactive dendrites. We conclude that the thalamic parafascicular nucleus indeed sends synaptic input to parvalbumin-immunoreactive striatal neurons. Parafascicular nucleus inputs to striatal parvalbumin-immunoreactive interneurons are sparse in comparison to other asymmetric inputs, most of which are likely to be of cortical origin. The synaptic profile of thalamostriatal inputs to parvalbumin-immunoreactive neurons and unlabeled elements is unchanged following neonatal decortication. This suggests that competitive interaction between developing thalamostriatal and corticostriatal projections is not a major mechanism determining synaptic input to striatal subpopulations.

Rapid urinary iodide test.

Assessment of iodine deficiency and monitoring of iodine supplementation programs demands rapid, simple and cost-effective methods for the determination of urinary iodide concentrations. We propose a rapid test based on the iodide-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine by peracetic acid/H2O2 to yield colored products. The color of the chemical reaction is compared with color categories of a pictogram corresponding to three ranges (<10, 10-30, and >>30 microg/100 mL) of iodide concentrations. The test is very easy to perform and does not require any instrumentation or apparatus. Sample preparation is simple and consists in the removal of interfering substances by disposable columns, 65 x 10.5 mm, packed with specifically prepared activated charcoal. For comparison with a reference method for measuring urinary iodide (HPLC), we determined the iodide concentrations of 370 random (untimed) urine samples from consecutive patients by both HPLC and the rapid test. The results obtained by both methods are in close agreement with respect to classification of the samples according to the above three ranges, with a maximum difference of <5% for each range. This rapid test is therefore very well suited to epidemiological surveys of iodine deficiency especially in developing countries.