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International Journal of Molecular... Jan 2024Mass photometry (MP) is a fast and simple analysis method for the determination of the proportions of subpopulations in an AAV sample. It is label-free and requires...
Mass photometry (MP) is a fast and simple analysis method for the determination of the proportions of subpopulations in an AAV sample. It is label-free and requires minimal sample volumes between 5-10 µL, which makes it a promising candidate over orthogonal techniques such as analytical ultracentrifugation (AUC), cryo-transmission electron microscopy (Cryo-TEM) or charge-detection mass spectrometry (CDMS). However, these methods are limited in their application to purified samples only. Here we developed a purification step based on single-domain monospecific antibody fragments immobilised on either a poly(styrene-divinylbenzene) resin or on magnetic beads prior to MP analysis that allows the quantification of empty, partially filled, full and overfull AAV vectors in crude cell extracts. This is aimed at identifying potentially promising harvest conditions that yield large numbers of filled AAV vectors during the early stages of the viral vector development platform, e.g., the type of transfection reagent used. Furthermore, we provide a direct comparison of the automated and manual handling of the mass photometer with respect to the quantities of AAV subspecies, molar mass of the capsid and payload, and highlight the differences between the "buffer-free" sample measurement and the "buffer-dilution" mode. In addition, we provide information on which candidates to use for calibration and demonstrate the limitations of the mass photometer with respect to the estimation of the capsid titer.
Topics: Cell Extracts; Dependovirus; Biotechnology; Calibration; Capsid Proteins; Photometry; Single-Domain Antibodies
PubMed: 38255912
DOI: 10.3390/ijms25020838 -
Ocular Immunology and Inflammation Nov 2023To evaluate the correlation between longitudinal changes in aqueous flare measured by laser flare photometer (LFP), best-corrected visual acuity (BCVA), and clinical...
PURPOSE
To evaluate the correlation between longitudinal changes in aqueous flare measured by laser flare photometer (LFP), best-corrected visual acuity (BCVA), and clinical grade using both Standardization of Uveitis Nomenclature (SUN) and modified SUN (MSUN) scales uveitis patients.
METHODS
Patients were classified according to both SUN and MSUN grading scales. LFP measurements were acquired (Kowa FM-700) at each visit. Mean change in LFP was assessed longitudinally, comparing with those in visual acuity, SUN, and MSUN grading scales.
RESULTS
Mean change in LFP was correlated to those in BCVA ( = .018), SUN scale ( < .001), and MSUN scale ( = .008). Cases within same initial SUN (0 and 1+) and MSUN (0.5+ and 1+) grades and different longitudinal flare prognosis (decreased/unchanged/increased) had significantly different initial LFP values (all < .05).
CONCLUSIONS
LFP measurement is beneficial in monitoring inflammatory activity. Cases of identical clinical flare scores with different clinical prognosis may be predicted by LFP.
Topics: Humans; Aqueous Humor; Uveitis, Anterior; Uveitis; Photometry; Lasers
PubMed: 36170559
DOI: 10.1080/09273948.2022.2123365 -
Environmental Science and Pollution... Nov 2023This study investigates the absorption behavior of natural dyes on silk fabric: madder roots (red dye) and amaltas (brown dye). The dyes were extracted under optimized...
This study investigates the absorption behavior of natural dyes on silk fabric: madder roots (red dye) and amaltas (brown dye). The dyes were extracted under optimized conditions in an acidic medium. Binary mixtures of these dyes in different proportions were employed to develop various shades. This research work has explored the impact of single as well as binary mixture of chemical mordants on the dyeing behavior of natural dyes on silk. Al (Alum), iron (Fe), and tannic acid (T.A.) were used as pre- and post-mordants. Different concentrations of the dye mixtures were used to achieve different shades. The absorption behavior of the binary mixture of natural dyes was assessed using a spectra photometer Colori-spectra SF600. The study found that the highest color strength was achieved when an acidic extract with a table salt concentration of 2 g/100 mL was applied to silk fabric at 65 °C for 55 min and subjected to microwave treatment for 6 min. The study revealed excellent results for the selected binary mixtures of chemical mordants, such as (Al + Fe), (Al + T.A.), and (Fe + T.A.), in comparison by employing single mordants such as (Al, Fe, and T.A.).
Topics: Coloring Agents; Silk; Textiles; Plants; Tannins
PubMed: 37840083
DOI: 10.1007/s11356-023-30162-y -
Journal of Veterinary Internal Medicine 2024Ascorbic acid (AA) is an antioxidant that might be beneficial for adjunctive treatment of sepsis in horses. The optimal dose and effects on oxidative status are unknown.
BACKGROUND
Ascorbic acid (AA) is an antioxidant that might be beneficial for adjunctive treatment of sepsis in horses. The optimal dose and effects on oxidative status are unknown.
HYPOTHESIS
Ascorbic acid administration will increase plasma AA concentrations and decrease determinants of reactive oxygen metabolites (dROM), basal and stimulant-induced intraerythrocytic reactive oxygen species (ROS) concentrations, and stimulant-induced neutrophil ROS production, and increase plasma antioxidant capacity (PAC) in a dose-dependent manner.
ANIMALS
Eight healthy horses.
METHODS
Randomized placebo-controlled crossover study. Each horse received 4 single-dose IV treatments including AA at 25, 50, and 100 mg/kg and saline (placebo) with each treatment separated by ≥1 week. Blood was collected at baseline, 2 and 6 hours for assessment of plasma dROM and PAC via photometer, intraerythrocytic ROS by flow cytometry, and stimulant-induced neutrophil ROS by a fluorometric assay. Plasma AA concentrations were measured by high-performance liquid chromatography/electrochemical detection.
RESULTS
Ascorbic acid at 100 mg/kg resulted in decreased dROM 2 hours after treatment (P = .03, 95% CI 5.51-121.2, point estimate 63.3). There was no effect of AA on basal or stimulant-induced intraerythrocytic ROS (P = .88, 95% CI -0.156 to 0.081, point estimate -0.037; P = .93, 95% CI -0.123 to 0.112, point estimate -0.006, respectively), basal or stimulant-induced neutrophil ROS (P ≥ .12, 95% CI -644.9 to 56.2, point estimate -294.4), or PAC (P ≥ .64, 95% CI -1567 to 463.4, point estimate -552.0) at any dose or timepoint. Plasma AA concentrations increased in a dose-dependent manner.
CONCLUSIONS AND CLINICAL IMPORTANCE
High-dose administration of AA might provide antioxidant benefits in horses.
Topics: Horses; Animals; Ascorbic Acid; Antioxidants; Reactive Oxygen Species; Cross-Over Studies; Oxidative Stress; Vitamins; Oxygen; Administration, Intravenous
PubMed: 37948618
DOI: 10.1111/jvim.16934 -
The Analyst Sep 2023The development of microfluidic systems for biological assays presents challenges, particularly in adapting traditional optical absorbance assays to smaller volumes or...
The development of microfluidic systems for biological assays presents challenges, particularly in adapting traditional optical absorbance assays to smaller volumes or to microfluidic formats. This often requires assay modification or translation to a fluorescence version, which can be impractical. To address this issue, our group has developed the μChopper device, which uses microfluidic droplet formation as a surrogate for an optical beam chopper, allowing for lock-in analysis and improved limits of detection with both absorbance and fluorescence optics without modifying the optical path length. Here, we have adapted the μChopper to low-cost optics using a light-emitting diode (LED) source and photodiode detector, and we have fabricated the pnuematically valved devices entirely by 3D printing instead of traditional photolithography. Using a hybrid device structure, fluidic channels were made in polydimethylsiloxane (PDMS) by moulding onto a 3D-printed master then bonding to a prefabricated thin layer, and the pneumatic layer was directly made of 3D-printed resin. This hybrid structure allowed an optical slit to be fabricated directly under fluidic channels, with the LED interfaced closely above the channel. Vacuum-operated, normally closed valves provided precise temporal control of droplet formation from 0.6 to 2.0 Hz. The system was validated against the standard plate reader format using a colorimetric fructosamine assay and by quantifying fructosamine in human serum from normal and diabetic patients, where strong correlation was shown. Showing a standard benefit of microfluidics in analysis, the device required 6.4-fold less serum volume for each assay. This μChopper device and lower cost optical system should be applicable to various absorbance based assays in low volumes, and the reliance on inexpensive 3D printers makes it more accessible to users without cleanroom facilities.
Topics: Humans; Microfluidic Analytical Techniques; Fructosamine; Microfluidics; Printing, Three-Dimensional; Software
PubMed: 37605899
DOI: 10.1039/d3an01149f -
Journal of Environmental Sciences... Oct 2023Black carbon (BC) aerosols in the atmosphere play a significant role in climate systems due to their strong ability to absorb solar radiation. The lifetime of BC depends...
Black carbon (BC) aerosols in the atmosphere play a significant role in climate systems due to their strong ability to absorb solar radiation. The lifetime of BC depends on atmospheric transport, aging and consequently on wet scavenging processes (in-cloud and below-cloud scavenging). In this study, sequential rainwater samples in eight rainfall events collected in 2 mm interval were measured by a tandem system including a single particle soot photometer (SP2) and a nebulizer. The results showed that the volume-weighted average (VWA) mass concentrations of refractory black carbon (rBC) in each rainfall event varied, ranging from 10.8 to 78.9 µg/L. The highest rBC concentrations in the rainwater samples typically occurred in the first fraction from individual rainfall events. The geometric mean median mass-equivalent diameter (MMD) decreased under precipitation, indicating that rBC with larger sizes was relatively aged and preferentially removed by wet scavenging. A positive correlation (R = 0.73) between the VWA mass concentrations of rBC in rainwater and that in ambient air suggested the important contribution of scavenging process. Additionally, the contributions of in-cloud and below-cloud scavenging were distinguished and accounted for 74% and 26% to wet scavenging, respectively. The scavenging ratio of rBC particles was estimated to be 0.06 on average. This study provides helpful information for better understanding the mechanism of rBC wet scavenging and reducing the uncertainty of numerical simulations of the climate effects of rBC.
Topics: Air Pollutants; Beijing; Soot; Aerosols; Carbon; Environmental Monitoring
PubMed: 37336608
DOI: 10.1016/j.jes.2022.07.039 -
Therapeutic Advances in Ophthalmology 2023Anthropometry facilitates the evaluation of risks associated with reduced macular pigment optical density (MPOD).
BACKGROUND
Anthropometry facilitates the evaluation of risks associated with reduced macular pigment optical density (MPOD).
OBJECTIVES
To investigate the predictors and anthropometric indices associated with MPOD in healthy adult in Arab population.
DESIGN
This is a cross-sectional study.
METHODS
The MPOD was measured at 0.5° from fovea using a heterochromatic flicker photometer. Healthy participants aged between 20 and 40 years were recruited. The study evaluated the following data of the participants: height, weight, body mass index, body fat percentage, basal metabolic rate, visceral fat level, muscle mass, bone mineral content, and percentage of protein and body water. The correlation between MPOD with anthropometrics and demographic data was evaluated using Spearman's correlation test. The differences among genders were investigated using the Mann-Whitney test. The smoking effect on MPOD was analyzed using the Friedman test.
RESULTS
In all, 143 participants were recruited. The median ± interquartile range was calculated for age (23 ± 4 years), visual acuity (0.00 ± 0.00 logMAR), and MPOD (0.41 ± 0.18). The average MPOD was higher in males than in females but it was not statistically significant ( > 0.05); on the other hand, they were statistically significantly different in most of the anthropometric data. A significant relationship was found between MPOD and percentage of body fat, protein, and body water ( = 0.30, < 0.05). The observed median MPOD value was higher in this study than that found in previous studies in white populations, but lower than that found in studies investigating Asian populations.
CONCLUSION
One of the most important risk factors of age-related macular degeneration is associated with a relative absence of macular pigment. This study brought into focus percentage of protein and body water for further studies as well as the well-established links with body fat and obesity. Unknown predictors of MPOD remain uncovered. The study also provided first report on normative values of MPOD for Arab population and confirmed the differences from other ethnicities.
PubMed: 37599800
DOI: 10.1177/25158414231189099 -
The Science of the Total Environment Jan 2024We propose a simple technique for microplastic detection based on their interaction with a hydrophilic and anionic fluorescent dye 1,3,6,8 pyrene tetrasulfonate (PTSA)....
We propose a simple technique for microplastic detection based on their interaction with a hydrophilic and anionic fluorescent dye 1,3,6,8 pyrene tetrasulfonate (PTSA). Synthetic microfibers derived from surgical face masks (an abundantly generated plastic waste post COVID) were considered as model microplastics. The interactions between microfibers and the dye were studied as a function of physiological parameters (pH, contact time and temperature), external agents, dye dosage and polymer variants. A pocket-sized photometer (by Lovibond Tintometer group) was employed for the detection and further validated using advanced equipment set-ups (fluorescence microscope, Fourier transform infrared spectroscopy and benchtop spectrofluorometer). Risk assessment studies were conducted on Artemia salina as a model organism. As a risk mitigation strategy, dye recovery followed by sunlight mediated degradation were performed. The detection study was performed in real water samples collected from fresh, estuarine and seawater samples spiked with microfibers. As an outcome, an optimized standard operational conditions were determined for the effective detection of synthetic microfibers. The data obtained could have scientific and industrial impact, in particular for experts working in the broad arena of clean water, who are specifically interested in developing cost-effective solutions for effective detection and biomonitoring of emerging pollutants.
Topics: Plastics; Water; Spectrometry, Fluorescence; Water Pollutants, Chemical; Microplastics; Staining and Labeling; Environmental Monitoring
PubMed: 37944615
DOI: 10.1016/j.scitotenv.2023.168144 -
Sensors (Basel, Switzerland) Sep 2023This paper analyzes the light-scattering standards currently used for calibration (verification) and systematic research in photo and spectrophotometry tools. The...
This paper analyzes the light-scattering standards currently used for calibration (verification) and systematic research in photo and spectrophotometry tools. The application specificities in studying the diffuse reflected and transmitted light during biomedical CCD photometry are considered. The advantages of a new class of photometers with non-spherical reflectors as ellipsoids of revolution truncated along the focal planes with the internal mirror surface are presented. The ellipsoid first focal plane is combined with the surface of the under-study media, and the second is optically coupled to the CCD image sensor plane. The principles of zone analysis of spatial distribution reproduced in photometric images on a CCD sensor are substantiated. The illuminance levels of photometric image areas in reflected and transmitted light from the thickness of the standard for the wavelength of laser radiation of 650 nm of different power was experimentally investigated. Polynomial dependences were obtained, and regression coefficients of the illuminance of the external and middle rings in photometric images for the reflected and transmitted light on the laser power were determined.
PubMed: 37765756
DOI: 10.3390/s23187700 -
ACS Omega Aug 2023Herein, we present the immobilization of a technical grade β-d-galactosidase on amino-functionalized microtiter plates. Afterward, we transferred the results to a...
Herein, we present the immobilization of a technical grade β-d-galactosidase on amino-functionalized microtiter plates. Afterward, we transferred the results to a resin-based approach. For the covalent binding of the enzyme, an amino-functionalized microtiter plate was prefunctionalized with 1,4-phenylendiisothiocyanate. The cleavage of the substrate 5-bromo-4-chloro-3-indoxyl-β-d-galactopyranoside (X-Gal) produces a deep blue dye, which was quantified in a microtiter plate reader at 595 nm. The maximum reaction rates and the Michaelis-Menten constant were calculated. In addition, the unwanted blue precipitate formed during the experiments could be minimized by optimizing the experiments. When transferring the immobilization method to Rink amide resin, -nitrophenyl-β-d-galactopyranoside was used as the substrate and the measurement was carried out in a photometer at 420 nm.
PubMed: 37546597
DOI: 10.1021/acsomega.3c03279