Evaluation of semi-quantitative colorimetric assays based on loop-mediated isothermal amplification indicators by using image analysis.

Colorimetric assays are some of the most convenient detection methods, creating discoloration in solutions that is visible to the naked eye. However, colorimetric reactions have some limitations regarding the variability in the color perception of individuals caused by factors such as color blindness, experience, and gender. Semi-quantitative chromatic analysis has been used as an alternative method to differentiate between two colors and accurately interpret the results from a numerical value, with high confidence. Therefore, we developed and determined the optimal model between Red-Green-Blue (RGB) and Commission Internationale de l'Eclairage (CIE) Lab color spaces to establish a semi-quantitative colorimetric assay via image analysis by the ImageJ program for loop-mediated isothermal amplification (LAMP), using the dyes malachite green and phenol red. The semi-quantitative colorimetric assays using the color distance values of the CIELab color space (ΔEab) were more suitable than those using the RGB color space (ΔERGB) for chromatic differentiation between positive and negative reactions in both indicator dyes, demonstrating the feasibility of this assay to be applied in the detection of a wide range of pathogens and infectious diseases.

Validation of the phenol red thread test in a Chinese population.

BACKGROUND: To investigate the validation of phenol red thread (PRT) test in a Chinese population by evaluating the intraobserver repeatability and interobserver reproducibility, determining correlations between the PRT test and other dry eye disease (DED) parameters including tear meniscus height (TMH) and Schirmer I test, and testing the accuracy of diagnosing DED when using the PRT test alone. METHODS: A total of 108 eyes were involved in this prospective and diagnostic study, and were divided into two groups (with and without DED). Each subject underwent a series of ocular surface examinations, including Ocular Surface Disease Index (OSDI) questionnaire, non-invasive tear breakup time (NIBUT), tear meniscus height (TMH) assessment, PRT test, fluorescein tear breakup time (FBUT), corneal fluorescein staining and Schirmer I test. RESULTS: In the experimental group and the control group, the intra-class correlation coefficients (ICCs) of the repeatability were 0.747 and 0.723, respectively (all P < 0.05). The ICCs of the reproducibility in both groups were 0.588 and 0.610, respectively (all P < 0.05). The PRT test correlated weakly with the Schirmer I test and the tear meniscus height, with Spearman coefficients of 0.385 and 0.306, respectively (all P < 0.05). The PRT test is available to diagnose DED, with an area under the curve of 0.806 and a Youden index of 0.556 at the cutoff point of 8.83 mm. CONCLUSIONS: The PRT test can provide patients a comfortable, timesaving and less irritating approach to screening and diagnosing DED compared to Schirmer I test.

New insights on the adsorption of phenol red dyes from synthetic wastewater using activated carbon/Fe2(MoO4)3.

Water shortage is considered as one of the main challenges of human life. A practical solution to this problem is the wastewater treatment. The removal of dyes from wastewaters has received considerable critical attention by researchers due to their high volume and toxicity. In the current research, the adsorption of phenol red dyes from synthetic wastewater using the activated carbon produced from Mespilus germanica modified with Fe2(MoO4)3 was studied. The proposed adsorbent was characterized by Fourier transform infrared (FTIR), X-ray diffraction (XRD), scanning electron microscope (SEM), energy dispersive X-ray analysis (EDX)/Map, Brunauer-Emmett-Teller (BET), and Raman techniques. The optimal adsorption operating parameters including pH, stirring rate, temperature, dosage of adsorbent, dye initial concentration, and contact time were 3, 500 rpm, 25 °C, 1 g/L, 10 mg/L, and 60 min, respectively. Furthermore, the successful regeneration of the adsorbent for 3 times, using methanol solution as a regeneration medium, denoted its capability in performing adsorption and desorption processes. Equilibrium studies showed that the adsorption of phenol red dyes by activated carbon (AC)/Fe2(MoO4)3 was desirable and physical and the experimental data were fitted well by the Freundlich model. In addition, the kinetic behavior of the current adsorption process was well described by the pseudo-second-order kinetic model, while thermodynamic calculations showed that the process was exothermic and spontaneous.

Clinical testing on SARS-CoV-2 swab samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP).

BACKGROUND: High cost of commercial RNA extraction kits limits the testing efficiency of SARS-CoV-2. Here, we developed a simple nucleic acid extraction method for the detection of SARS-CoV-2 directly from nasopharyngeal swab samples. METHODS: A pH sensitive dye was used as the end point detection method. The obvious colour changes between positive and negative reactions eliminates the need of other equipment. RESULTS: Clinical testing using 260 samples showed 92.7% sensitivity (95% CI 87.3-96.3%) and 93.6% specificity (95% CI 87.3-97.4%) of RT-LAMP. CONCLUSIONS: The simple RNA extraction method minimizes the need for any extensive laboratory set-up. We suggest combining this simple nucleic acid extraction method and RT-LAMP technology as the point-of care diagnostic tool.

Closed-tube field-deployable loop-mediated isothermal amplification (LAMP) assay based on spore wall protein (SWP) for the visual detection of Enterocytozoon hepatopenaei (EHP).

Hepatopancreatic microsporidiosis (HPM) is an infectious shrimp disease caused by the microsporidian Enterocytozoon hepatopenaei (EHP). In recent years, the widespread occurrence of EHP poses a significant challenge to the shrimp aquaculture industry. Early, rapid and accurate diagnosis of EHP infection is very much essential for the control of HPM crop-related losses. Loop-mediated isothermal amplification (LAMP) is a robust, sensitive, cost-effective disease diagnostic technique. Here, we demonstrate an improved, simple, closed-tube, colorimetric EHP LAMP diagnostic assay. LAMP assay was illustrated with the specific EHP spore wall protein (SWP) gene primers. Naked eye visual detection of LAMP amplicons was achieved using Hydroxy naphthol blue (HNB) or Phenol red dye without opening the tubes. This LAMP assay is efficient in detecting the EHP pathogen in all clinical samples include shrimp hepatopancreas, FTA card samples, feces, pond water, and soil. Also, the elution of EHP DNA from FTA cards was demonstrated within 17 min using a simple dry bath. In clinical evaluation, the visual LAMP assay established 100% diagnostic sensitivity and 100% diagnostic specificity. The visual LAMP assay is rapid, can detect the EHP pathogen within 40 min using a simple dry bath, and does not require any expensive instruments and technical proficiency. In conclusion, this visual LAMP protocol is a user-friendly, specific assay that can be conceivably operated at the farm-site/ resource-limited settings by the farmer himself with simple equipment.

A Ratiometric Optical Dual Sensor for the Simultaneous Detection of Oxygen and Carbon Dioxide.

Simultaneous detection of carbon dioxide (CO2) and oxygen (O2) has attracted considerable interest since CO2 and O2 play key roles in various industrial and domestic applications. In this study, a new approach based on a fluorescence ratiometric referencing method was reported to develop an optical dual sensor where platinum (II) meso-tetrakis(pentafluorophenyl)porphyrin (PtTFPP) complex used as the O2-sensitive dye, CdSe/ZnS quantum dots (QDs) combined with phenol red used as the CO2-sensitive dye, and CdSe/ZnS QDs used as the reference dye for the simultaneous detection of O2 and CO2. All the dyes were immobilized in a gas-permeable matrix poly (isobutyl methacrylate) (PolyIBM) and subjected to excitation using a 380 nm LED. The as-obtained distinct fluorescence spectral intensities were alternately exposed to analyte gases to observe changes in the fluorescence intensity. In the presence of O2, the fluorescence intensity of the Pt (II) complex was considerably quenched, while in the presence of CO2, the fluorescence intensity of QDs was increased. The corresponding ratiometric sensitivities of the optical dual sensor for O2 and CO2 were approximately 13 and 144, respectively. In addition, the response and recovery for O2 and CO2 were calculated to be 10 s/35 s and 20 s/60 s, respectively. Thus, a ratiometric optical dual gas sensor for the simultaneous detection of O2 and CO2 was successfully developed. Effects of spurious fluctuations in the intensity of external and excitation sources were suppressed by the ratiometric sensing approach.

Nanoparticulate tablet dosage form of lisofylline-linoleic acid conjugate for type 1 diabetes: in situ single-pass intestinal perfusion (SPIP) studies and pharmacokinetics in rat.

Lisofylline (LSF) is an anti-inflammatory molecule with high aqueous solubility and rapid metabolic interconversion to its parent drug, pentoxifylline (PTX) resulting in very poor pharmacokinetic (PK) parameters, necessitating high dose and dosing frequency. In the present study, we resolved the physicochemical and pharmacokinetic limitations associated with LSF and designed its oral dosage form as a tablet for effective treatment in type 1 diabetes (T1D). Self-assembling polymeric micelles of LSF (lisofylline-linoleic acid polymeric micelles (LSF-LA PLM)) were optimized for scale-up (6 g batch size) and lyophilized followed by compression into tablets. Powder blend and tablets were evaluated as per USP. LSF-LA PLM tablet so formed was evaluated for in vitro release in simulated biological fluids (with enzymes) and for cell viability in MIN-6 cells. LSF-LA PLM in tablet formulation was further evaluated for intestinal permeability (in situ) along with LSF and LSF-LA self-assembled micelles (SM) as controls in a rat model using single-pass intestinal perfusion (SPIP) study. SPIP studies revealed 1.8-fold higher oral absorption of LSF-LA from LSF-LA PLM as compared to LSF-LA SM and ~5.9-fold higher than LSF (alone) solution. Pharmacokinetic studies of LSF-LA PLM tablet showed greater Cmax than LSF, LSF-LA, and LSF-LA PLM. Designed facile LSF-LA PLM tablet dosage form has potential for an immediate decrease in the postprandial glucose levels in patients of T1D.

Cyanuric Acid Biodegradation via Biuret: Physiology, Taxonomy, and Geospatial Distribution.

Cyanuric acid is an industrial chemical produced during the biodegradation of s-triazine pesticides. The biodegradation of cyanuric acid has been elucidated using a single model system, Pseudomonas sp. strain ADP, in which cyanuric acid hydrolase (AtzD) opens the s-triazine ring and AtzEG deaminates the ring-opened product. A significant question remains as to whether the metabolic pathway found in Pseudomonas sp. ADP is the exception or the rule in bacterial genomes globally. Here, we show that most bacteria utilize a different pathway, metabolizing cyanuric acid via biuret. The new pathway was determined by reconstituting the pathway in vitro with purified enzymes and by mining more than 250,000 genomes and metagenomes. We isolated soil bacteria that grow on cyanuric acid as a sole nitrogen source and showed that the genome from a Herbaspirillum strain had a canonical cyanuric acid hydrolase gene but different flanking genes. The flanking gene trtB encoded an enzyme that we show catalyzed the decarboxylation of the cyanuric acid hydrolase product, carboxybiuret. The reaction generated biuret, a pathway intermediate further transformed by biuret hydrolase (BiuH). The prevalence of the newly defined pathway was determined by cooccurrence analysis of cyanuric acid hydrolase genes and flanking genes. Here, we show the biuret pathway was more than 1 order of magnitude more prevalent than the original Pseudomonas sp. ADP pathway. Mining a database of over 40,000 bacterial isolates with precise geospatial metadata showed that bacteria with concurrent cyanuric acid and biuret hydrolase genes were distributed throughout the United States.IMPORTANCE Cyanuric acid is produced naturally as a contaminant in urea fertilizer, and it is used as a chlorine stabilizer in swimming pools. Cyanuric acid-degrading bacteria are used commercially in removing cyanuric acid from pool water when it exceeds desired levels. The total volume of cyanuric acid produced annually exceeds 200 million kilograms, most of which enters the natural environment. In this context, it is important to have a global understanding of cyanuric acid biodegradation by microbial communities in natural and engineered systems. Current knowledge of cyanuric acid metabolism largely derives from studies on the enzymes from a single model organism, Pseudomonas sp. ADP. In this study, we obtained and studied new microbes and discovered a previously unknown cyanuric acid degradation pathway. The new pathway identified here was found to be much more prevalent than the pathway previously established for Pseudomonas sp. ADP. In addition, the types of environment, taxonomic prevalences, and geospatial distributions of the different cyanuric acid degradation pathways are described here.

Paper chip-based colorimetric assay for detection of Salmonella typhimurium by combining aptamer-modified Fe3O4@Ag nanoprobes and urease activity inhibition.

A rapid and sensitive colorimetric assay is described for Salmonella typhimurium (S. typhimurium) detection using urea/phenol red impregnated test paper. Aptamer-modified Fe3O4@Ag multifunctional hybrid nanoprobes (apt-Fe3O4@Ag NPs) were used to specifically captured S. typhimurium; the nanoprobes were quickly etched by H2O2 to form Ag+. The generated Ag+ can inhibit the urease-catalyzed hydrolysis reaction of urea to produce NH4+. Consequently, the as-prepared test paper displayed a yellow color. In the presence of S. typhimurium, the target bacteria can cause aggregation of apt-Fe3O4@Ag NPs, and the deposited Ag on the nanoprobe's surface is shielded against H2O2-induced oxidative decomposition leading to reduced Ag+ production. The catalytic activity of urease cannot be inhibited completely by inadequate amount of Ag+. An obvious color change from yellow to pink can be monitored directly using our test paper as a result of increased NH4+. The entire assay procedure could be completed within 1 h. A limit of detection of 48 cfu/mL is achieved with a linear range of 1 × 102 to 1 × 106 cfu/mL. The recoveries of S. typhimurium spiked in pure milk samples were 92.48-94.05%. Graphical abstract Schematic diagram of the proposed colorimetric assay for S. typhimurium detection based on etching of bifunctional apt-Fe3O4@Ag NPs and inhibiting catalytic activity of urease by Ag+. A color change from yellow to pink can be observed and correlated to the concentration of S. typhimurium.

Ophthalmic examination, biometry, and histologic findings in captive inland bearded dragons (Pogona vitticeps).

OBJECTIVE: To report ophthalmic examination, biometry, phenol red thread test (PRTT), intraocular pressure (IOP), and histologic findings from a private collection of inland bearded dragons (Pogona vitticeps). ANIMALS STUDIED: Fourteen inland bearded dragons. PROCEDURES: Complete ophthalmic examinations were performed on all animals, including slit-lamp biomicroscopy, indirect ophthalmoscopy, fluorescein stain, phenol red thread test, and rebound tonometry. B-mode ultrasonography was used to measure anterior chamber depth, axial lens thickness, vitreal chamber depth, and axial globe length. Horizontal corneal diameter was estimated using ImageJ software. Histologic assessment was obtained for one of the bearded dragons that died following the study period. RESULTS: The median PRTT value was 7.27 mm/15 seconds. Mean IOP was 6.29 ± 1.60 mm Hg and 2.14 ± 1.37 mm Hg using the dog and undefined calibration settings, respectively. Median axial globe length was 11.75 mm. Mean anterior chamber depth and mean lens thickness were 2.06 ± 0.35 mm and 3.38 ± 0.45 mm, respectively. Median vitreal chamber depth was 6.79 mm. Mean horizontal corneal diameter was 5.138 ± 0.346 mm. Two distinct ocular phenotypes were observed, with two of the bearded dragons having corneal globosa, deep anterior chambers, and tufts of iridal vessels and fibrillar material extending into the anterior chamber. CONCLUSIONS: The ultrasound biometry, PRTT, and rebound tonometry results may serve as a guideline for ophthalmic parameters in healthy bearded dragons. Examination and testing of greater numbers of animals are necessary to establish true reference ranges and determine if the observed ocular phenotypes represent normal variants or pathologic changes.

Environmental relative humidity affects Schirmer tear test results in normal dogs.

OBJECTIVE: To investigate the effects of environmental temperature and relative humidity on tear test results in dogs. ANIMALS STUDIED: 22 normal dogs. PROCEDURES: Routine Schirmer tear tests (STT1) and phenol red thread tests (PRTT) were administered to normal dogs in four environmental conditions with different relative humidity (RH) and temperature (T). Environmental conditions (mean ± SD) included the following: normal indoor (RH = 49 ± 4%, T = 72 ± 2°F), outdoor (RH = 62 ± 11%, T = 85 ± 6°F), indoor high humidity (RH = 85 ± 6%, T = 71 ± 1°F), and indoor low humidity (RH = 39 ± 2%, T = 76 ± 1°F). ANCOVA models were used to assess for significant associations between tear test readings and RH/T (P < .05). RESULTS: A significant, direct positive relationship between RH and STT1 results was detected (y = 0.04909*X + 19.11, P = .006) but not between T and STT1. There was no significant relationship between PRTT and RH or T. CONCLUSIONS: These results suggest that STT1 but not PRTT is affected by RH and that T does not influence STT1 or PRTT results in normal dogs. RH is unlikely, however, to cause clinically significant effects on STT1 readings in normal dogs when performed in average indoor conditions. Future studies should investigate the influence of RH on STT1 readings obtained in dogs with keratoconjunctivitis sicca.

Ophthalmic findings in cinereous vultures (Aegypius monachus).

OBJECTIVE: The aim of the present study was to provide ophthalmic reference values under normal physiological conditions for Aegypius monachus (cinereous vulture). PROCEDURES: Thirty-two eyes of sixteen adult captive cinereous vultures were used for this study. Tear tests and tonometry in conscious and anesthetized states, neuro-ophthalmic tests, measurement of corneal diameter, slit-lamp biomicroscopy, ophthalmoscopy, and funduscopy were performed. RESULTS: Schirmer tear test (STT) value was 11.4 ± 2.6 and 11.5 ± 2.8 mm/min in the right (OD) and left eye (OS), respectively. Phenol red thread test (PRT) values were 22.3 ± 2.1 mm/15 s OD and 22.8 ± 3.0 mm/15 s OS. The results showed a strong correlation between STT and PRT in both eyes. Intraocular pressure (IOP) values were 32.8 ± 6.9 mm Hg OD and 31.9 ± 7.1 mm Hg OS with TonoVet and 20.7 ± 4.5 mm Hg OD and 19.5 ± 4.1 mm Hg OS with Tono-Pen. There were significant differences in IOPs between rebound and applanation tonometry in both OD and OS. Tear production and IOP values showed significant reductions with general anesthesia in both tear tests and both tonometry (P < .001). Horizontal corneal diameter (mm) was 15.56 ± 0.96 OD and 15.56 ± 0.96 OS. Vertical diameter (mm) was 14.13 ± 0.96 OD and 14.06 ± 1.06 OS. The horizontal diameter was significantly longer than vertical diameter (P < .001). CONCLUSIONS: Ocular morphologic information and normal reference range values for various ophthalmic measurements were obtained in clinically healthy cinereous vultures, which can facilitate accurate diagnosis and better management of ophthalmic diseases in cinereous vultures.

Normative ocular data for juvenile and adult Japanese quail (Coturnix japonica).

OBJECTIVE: To obtain normative ocular data for Japanese quail as they mature from juveniles to adults. ANIMALS STUDIED: Twenty-six captive Japanese quail comprising thirteen males and thirteen females, free of ocular disease, were included in the study. PROCEDURES: Ophthalmic reference values were measured in both eyes at 1 and 5 months of age. A complete ophthalmic examination was performed, including neuro-ophthalmic reflexes, slit lamp biomicroscopy, phenol red thread test (PRTT), rebound tonometry, fluorescein staining, horizontal corneal diameter measurement, indirect ophthalmoscopy, and ocular ultrasound biometry. Ultrasound biomicroscopy measurements of axial globe length, lens thickness, vitreal chamber length, and pecten length were recorded. The depth of the anterior chamber was calculated by subtracting the lens thickness and vitreous length from the axial globe length. Measures of association and descriptive statistics were analyzed using STATA-14 and STATA-15. RESULTS: Juvenile and adult females were heavier than age-matched males. Weight, intraocular pressure, horizontal corneal diameter, axial globe length, and lens thickness measurements increased with age. No statistically significant differences were found in the remainder of measurements among individuals in different sex or age-groups. CONCLUSIONS: This work provides reference values and clinical findings that can be used in future research on quail and ocular disease.

Indicator dye based screening of glutaminase free L-asparaginase producer and kinetic evaluation of enzyme production process.

Several soil isolates from 1 g of soil sample were isolated and screened for the production of L-asparaginase. Primary screening was performed using rapid plate assay; dye indicator studies were conducted, and phenol red with 0.005% concentration was found to be optimum. The secondary screening was carried out using the Nesslerization method. The bacteria screened for L-asparaginase production with no glutaminase activity was identified as Bacillus subtilis. Crude L-asparaginase enzyme was partially purified 1.57 folds of purity and 110 U/mg of specific activity. The glutaminase-free L-asparaginase activity was also confirmed using LC-MS analysis. The presence of mass peaks at 147.0 in the reaction mixture suggested an absence of glutaminase activity. An optimized medium obtained comprised of Dextrose 1.5 g/L, K2HPO4 1.2 g/L, L-asparagine 15 g/L, and Tryptone 5 g/L. The highest L-asparaginase activity was observed at 6.0 pH and 30 °C. Kinetic parameters associated with biomass and L-asparaginase production were also studied. The computed values were µm 0.104 h-1, Xm 6g/L P0 1.7U/mL Pm 8.2 U/mL YX/S 4 g-cell/g-glucose µPm 0.35 h-1 qp 5.46 U/g/h YP/x 13.6667 U/g-cell. The novel bacterial isolates showed promise as a potential glutaminase-free L-asparaginase producer, which can prove to be of industrial applications.

Synthesis and Evaluation of PEG-PR for Water Flux Correction in an In Situ Rat Perfusion Model.

Phenol red (PR) is a widely used marker for water flux correction in studies of in situ perfusion, in which intestinal absorption usually leads to the underestimation of results. In this paper, we propose a novel marker polyethylene glycol (PEG)-PR (i.e., PR modified by PEGylation) with less permeability and evaluate its application in an in situ perfusion model in rats. PEG-PR was synthesized by the chemical conjunction of polyethylene glycol-4k/5k (PEG-4k/5k) and PR. The synthesized PEG-PR was then characterized using 1H-NMR, 13C-NMR, ultraviolet (UV), X-ray diffraction (XRD), and differential scanning calorimetry (DSC) analyses. The low permeability of PEG-PR was assessed using everted gut sac (EGS) methods. The apparent permeability coefficients (Papp, 3-8 × 10-7 cm/s) of PEG4k/5k-PR exhibited a nearly 15-fold reduction compared to that of PR. The different concentrations of PEG4k/5k-PR did not contribute to the Papp value or cumulative permeable percentage (about 0.02-0.06%). Furthermore, the larger molecular weight due to PEGylation (PEG5k-PR) enhanced the nonabsorbable effect. To evaluate the potential application of the novel marker, atenolol, ketoprofen, and metoprolol, which represent various biopharmaceutics classification system (BCS) classes, were selected as model drugs for the recirculation perfusion method. The water flux corrected by PEG4k/5k-PR reflected the accuracy due to the nonabsorbable effect, while the effective intestinal membrane permeability (Peff) of atenolol corrected by PEG4k/5k-PR showed a statistically significant increase (p < 0.05) in different intestinal segments. In conclusion, PEG-PR is a promising marker for the permeability estimation when using the in situ perfusion model in rats.

Ocular Examinations and Investigation of Intraocular Pressure, Tear Production, Central Corneal Thickness, and Corneal Touch Threshold in a Captive Flock of Atlantic Puffins (Fratercula arctica).

Ocular examinations were completed on a group of 10 Atlantic puffins (Fratercula arctica), 5 males and 5 females that ranged in age from 8 months to older than 30 years. The exams consisted of intraocular pressure/rebound tonometry, tear production/phenol red thread test, central corneal thickness/ultrasound pachymetry, and corneal sensitivity/esthesiometry. On ocular examination, there were no corneal abnormalities observed. Bilateral cataracts were diagnosed in 8 puffins, 6 of which were considered incipient, focal subcapsular opacities. One bird had hypermature cataracts and was removed from the study and excluded from data analysis; the other birds had no evidence of ophthalmic pathology that would interfere with diagnostic results (n = 9). All results for 9 birds were included in the study, with the exception of 1 puffin's tear production, which was too low for accurate assessment and was excluded from data analysis. There were no significant differences between right and left eye measurements for intraocular pressure, corneal thickness, and corneal sensitivity. The median intraocular pressure for both eyes (OU) was 13 mm Hg with an interquartile range [IQR] of 12-15 mm Hg. The median corneal thickness OU was 241 µm, IQR 233-248 µm. The median corneal sensitivity OU was 1.13 cm, IQR 0.81-1.50 cm. There was a significant difference between right and left eye measurements for tear production (right eye median, 7.5 mm/15 s, IQR 6.5-9.3 mm/15 s; and left eye median, 5.0 mm/15 s, IQR 4.0-7.3 mm/15 s) (P= .03), with the right eye producing more tears than the left. However, 1 puffin was determined to be an outlier, and when removed, there was no longer a significant difference (OU median, 7.0 mm/15 s, IQR 4.6-8.0 mm/15 s) (P = .38). There was no significant difference between sex and intraocular pressure, tear production, and corneal sensitivity. However, there was a significant difference between sex and corneal thickness (P = .02), with males (left eye median, 249 µm, IQR 241-249 µm) having thicker corneas than females (left eye median, 236 µm, 234-238 µm). Although sample size precluded statistical testing, there appeared to be an association between opacities and increasing age. There were no associations between age and intraocular pressure, tear production, or corneal thickness. There was a moderate correlation between age and corneal sensitivity, with older birds showing decreased corneal sensitivity (r = -0.57). Although the sample size of 9 birds was small, these findings provide preliminary ranges for ocular parameters of Atlantic puffins.

Role of phenol red in the stabilization of the Sabin type 2 inactivated polio vaccine at various pH values.

To analyze the effects of phenol red at various pH values on the Sabin type 2 inactivated polio vaccine (sIPV2), several biophysical techniques were used to evaluate the particle size and capsid protein for conformation. sIPV2's size was assessed via transmission electron microscopy and dynamic light scattering. The effects of various pH values (from 4.0 to 7.0) on the biophysical characters of sIPV2 particles in solution were determined by dynamic light scattering and zeta potential. The results clearly indicated that aggregation and instability occurred in the solution of sIPV2 particles at a pH of 6.0. Under similar conditions, by dynamic light scattering and zeta potential, the virus particles in solution showed more dispersion and were stable with the addition of 0.05 mM phenol red. According to circular dichroism and intrinsic tryptophan fluorescence data, it was observed that the secondary and tertiary structures of the sIPV2 particles were more stable with the protection of phenol red. At a pH below 6.0, the sIPV2 solution with phenol red had more D-antigen content, which was confirmed by enzyme-linked immunosorbent assay and rat experiments. These results strongly suggested that phenol red improved the pH stability of the sIPV2. The study indicated the potential of phenol red in preserving vaccine potency of the sIPV2 at various pH values.

A novel strip meniscometry method for measuring aqueous tear volume in dogs: Clinical correlations with the Schirmer tear and phenol red thread tests.

OBJECTIVES: The strip meniscometry test (SMT) is a novel method for quantitative measurement of tear volume with only five seconds. We aimed to evaluate clinical correlations of SMT with the gold standard Schirmer tear test (STT) and phenol red thread test (PRT) in dogs, including normal and tear-deficient eyes. ANIMALS STUDIED: Left eyes from 621 outpatient dogs with and without ocular disorders were evaluated. PROCEDURES: Each subject underwent SMT, PRT, and STT without topical anesthesia in the described order with five-minute intervals. The total population was divided into four groups by classifying tear deficiency severity based on STT results: "severe" (0-5 mm/min), "moderate" (6-10 mm/min), "subclinical" (11-14 mm/min), and "normal" (15 or more mm/min). RESULTS: The strongest correlation coefficient was found between SMT-STT (0.676), followed by PRT-STT (0.637) and SMT-PRT (0.600) pairs. Mean(SD) scores of SMT, PRT, and STT in total population were 9.47 (4.08) mm/5 s, 33.30 (8.52) mm/15 s, and 16.47 (7.01) mm/min. Significant differences were found among STT-classified groups, both using SMT and PRT results. Receiver operating characteristic (ROC) curves revealed that SMT better agreed with STT than PRT; agreement increased with increasing STT severity. A cutoff for SMT was identified at 10 mm/5 s to discriminate normal eyes from tear-deficient eyes, yielding high sensitivities and acceptable specificities. CONCLUSIONS: SMT could be superior to PRT for discriminating tear-deficient eyes. The high sensitivity of SMT could be useful as an initial diagnostic tool to rule out normal eyes with the short testing time.

Bare eye detection of Hg(II) ions based on enzyme inhibition and using mercaptoethanol as a reagent to improve selectivity.

The authors describe a colorimetric method for the determination of Hg2+ ions based on the inhibition of the activity of the enzyme urease. The pH value of solution increases when urease hydrolyzes urea, which can be visualized by adding a pH indicator such as Phenol Red (PhR). Mercaptoethanol as a typical thiol is added to the system to improve selectivity because it binds metal ions and then - unlike the Hg2+ mercaptoethanol complex - does not inhibit urease. Hence, the color of the pH indicator PhR turns from yellow to pink as the solution becomes alkaline. The Hg2+ mercaptoethanol complex, in contrast, strongly inhibits urease and the color of the solution remains yellow. The findings were used to design a photometric assay based on the measurement of the ratio of absorptions of PhR at 558 nm and 430 nm. It has a linear response over the 25 to 40 nM Hg2+ concentration range and a 5 nM detection limit. This is well below the guideline values of Hg2+ specified by the US Environmental Protection Agency and the World Health Organization for drinking water (10 nM and 30 nM, respectively). The method was employed to the determination of Hg2+ in water samples spiked with 10 nM levels of Hg2+ where color changes still can be observed visually. Graphical Abstract Schematic presentation of a colorimetric method for the ultrasensitive detection of Hg2+ based on the inhibition of urease activity. Mercaptoethanol is used to improve the selectivity. Even at Hg2+ concentrations as low as 5 nM, the color change still can be easily observed by bare eyes.

[Validation of in situ single pass perfusion model based on P-gp].

To validate in situ rats intestinal single pass perfusion model based on P-glycoprotein (P-gp). Firstly, phenol red perfusion was carried out to verify the close connection structure of intestinal epithelial cells, and the integrity of the intestinal epithelium, with a gravimetric method for correcting water flux. The level of phenol red was determined by high performance liquid chromatography (HPLC) both before and after perfusion. Secondly, the positive drug digoxin specified by FDA was used to validate the model. After different mass concentrations of verapamil were given in the rats, the absorption parameters of digoxin in ileum of rats were observed and compared. The results showed that the phenol red was absorbed in rats ileum segment, with an effective permeability coefficient of (1.09±0.62)×10 ⁻6 cm•s ⁻¹. The experiment results indicated that the close connection structure of intestinal epithelial cells was normal, and the integrity of the intestinal epithelium was maintained well. In digoxin perfusion experiment, in case no verapamil was given, digoxin showed certain degree of absorption in rat ileum, with an effective permeability coefficient (Peff) of (1.07±0.59)×10 ⁻5 cm•s ⁻¹; after mass concentrations of 0.01,0.1 mmol•L ⁻¹ verapamil were given, the absorption of digoxin was on the rise in rat ileum, with an effective permeability coefficient Peff of (1.58±0.69)×10 ⁻5, (3.28±0.95)×10 ⁻5 cm•s ⁻¹ respectively (P<0.05). Digoxin perfusion experiment verified that P-gp expression in small intestine epithelium was intact and can be used in the research of P-gp efflux transporter.