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Differential scanning fluorimetry (DSF) is a method to determine the apparent melting temperature (Tma) of a purified protein. In DSF, the raw unfolding curves from which Tma is calculated vary widely in shape and complexity. However, the tools available for calculating Tma are only compatible with the simplest of DSF curves, hindering many otherwise straightforward applications of the technology. To overcome this limitation, we designed new mathematical models for Tma calculation that accommodate common forms of variation in DSF curves, including the number of transitions, the presence of high initial signal, and temperature-dependent signal decay. When tested these models against DSFbase, an open-source database of 6235 raw, real-life DSF curves, these models outperformed the existing standard approaches of sigmoid fitting and maximum of the first derivative. To make these models accessible, we created an open-source software and website, DSFworld (https://gestwickilab.shinyapps.io/dsfworld/). In addition to these improved fitting capabilities, DSFworld also includes features that overcome the practical limitations of many analysis workflows, including automatic reformatting of raw data exported from common qPCR instruments, labeling of data based on experimental variables, and flexible interactive plotting. We hope that DSFworld will enable more streamlined and accurate calculation of Tma values for DSF experiments.

Aim: The aim of this study is to Quantify sialic acid for oral cancer diagnosis using different method using densitometric analysis. Background: Early detection of OSCC is critical for improving survival rates and reducing morbidity, which has prompted researchers to look for potential biomarkers to aid in early diagnosis. Saliva is an excellent diagnostic tool due to its noninvasive nature. Sialic acid levels in saliva have been found to be higher in cancer patients. Hypersialylation is associated with poor prognosis and accelerated cancer progression, as well as immune escape, tumor growth and spread, angiogenesis, and resistance to apoptosis and treatment. This study addresses the imperative need for adaptive, effective, and non-invasive strategies in the diagnosis and treatment of oral cancer, emphasising the importance of early detection for improved prognosis. This study introduces various methods for quantify levels of sialic acid in saliva, including resorcinol and orcinol assays, using colorimetric and densitometric assays. Material and Method: In this study, sialic acid detection methods have been standardized using standards as well as spent media from cancer cell lines to develop noninvasive diagnostic tools for early detection. Two methods used for the detection of sialic acid Resorcinal and Orcinol method by densitometric analysis using Image J software. Results: The study has successfully quantified sialic acid from spent media and saliva using resorcinol and orcinol method by densitometric analysis. Conclusion: In this study we aim to develop a reliable and cost-effective method for quantification of sialic acid using Image j software. The approach presents a non-invasive and efficient alternative for early cancer diagnosis showcasing its potential impact on advancing diagnostic technologies.

Modification of 2-acetyl-3H-benzo[f]chromen-3-one and 2-[(2E)-3-phenylprop-2-enoyl]-3H-benzo[f]chromen-3-one has been performed for the first time via the reaction with indole. The resulting benzo-[f]coumarins have been found to exhibit antioxidant properties in model systems: they react with hydrogen peroxide and sodium hypochlorite and regulate redox state of rat C6 glioma cells, which is reflected in reduced concentration of intracellular hydrogen peroxide and increased level of reduced glutathione. The synthesized compounds in the presence of exogenous hydrogen peroxide exert a protective effect on the cells, acting as antioxidants and restoring the redox balance. 2-[3-(1H-Indol-3-yl)-3-phenylpropanoyl]-3H-benzo[f]khromen-3-one at a micromolar concentrations inhibited the rat C6 glioma cell proliferation by 25–35%. [ABSTRACT FROM AUTHOR]

Dual-emissive fluorescence probes were designed by integrating porphyrin into the frameworks of UiO-66 for ratiometric fluorescence sensing of amoxicillin (AMX). Porphyrin integrated UiO-66 showed dual emission in the blue and red region. AMX resulted in the quenching of blue fluorescence component, attributable to the charge neutralization and hydrogen bonds induced energy transfer. AMX was detected using (F438/F654) as output signals. Two linear relationships were observed (from 10 to 1000 nM and 1 to 100 µM), with a limit of detection of 27 nM. The porphyrin integrated UiO-66 probe was used to detect AMX in practical samples. This work widens the road for the development of dual/multiple emissive fluorescence sensors for analytical applications, providing materials and theoretical supporting for food, environmental, and human safety. [ABSTRACT FROM AUTHOR]

When approving plant protection products, exposure data are required for risk analysis. Exposure data can be collected for various exposure pathways, such as dermal, inhalation or ground sediment. For measuring exposures, pyranine, a fluorescent dye, and a collector can be used. However, the choice of collector material depends on the specific exposure pathway. This study aims to determine the most suitable collector, in combination with the tracer pyranine, for recording exposure through different pathways in practical trials. Seven different collectors (Tyvek®, labels, plastic patches, paper patches, nylon filters, fibreglass filters, petri dishes) were subjected to laboratory and field tests to assess various quality parameters. Blank values, recovery rates, storability, and fluorescence degradation under UV-radiation were measured. Based on the results, a matrix was created summarizing which collector might be best suited to capture each exposure pathway. Almost all collectors demonstrated high recovery rates (Tyvek® 100%; labels 100%; plastic patches 100%; paper patches 100%; nylon filters 95%; fiberglass filters 60.9%) as well as good storability. Furthermore, all plastic-based collectors (labels, Tyvek®, plastic patches, petri dishes) showed a very good recovery rate above 95% when exposed to UV-radiation. However, nylon filters were not suitable for utilization under field conditions due to the rapid degradation of fluorescence under UV-radiation (recovery rate: 20–56%). Nevertheless, nylon filters showed stable recoveries under protected conditions and can be used to assess inhalation exposures under these conditions. Fibreglass filters, with recovery rates of 84–86%, were well-suited for testing inhalative exposures outdoors when a correction factor was applied. Tyvek® was the most suitable material for detecting total dermal exposure under field conditions. This is due to its quality characteristics and availability in a variety of sizes. Finally, petri dishes were ideal for collecting ground sediments. [ABSTRACT FROM AUTHOR]

Aim: The aim of this study is to Quantify sialic acid for oral cancer diagnosis using different method using densitometric analysis. Background: Early detection of OSCC is critical for improving survival rates and reducing morbidity, which has prompted researchers to look for potential biomarkers to aid in early diagnosis. Saliva is an excellent diagnostic tool due to its noninvasive nature. Sialic acid levels in saliva have been found to be higher in cancer patients. Hypersialylation is associated with poor prognosis and accelerated cancer progression, as well as immune escape, tumor growth and spread, angiogenesis, and resistance to apoptosis and treatment. This study addresses the imperative need for adaptive, effective, and non-invasive strategies in the diagnosis and treatment of oral cancer, emphasising the importance of early detection for improved prognosis. This study introduces various methods for quantify levels of sialic acid in saliva, including resorcinol and orcinol assays, using colorimetric and densitometric assays. Material and Method: In this study, sialic acid detection methods have been standardized using standards as well as spent media from cancer cell lines to develop noninvasive diagnostic tools for early detection. Two methods used for the detection of sialic acid Resorcinal and Orcinol method by densitometric analysis using Image J software. Results: The study has successfully quantified sialic acid from spent media and saliva using resorcinol and orcinol method by densitometric analysis. Conclusion: In this study we aim to develop a reliable and cost-effective method for quantification of sialic acid using Image j software. The approach presents a noninvasive and efficient alternative for early cancer diagnosis showcasing its potential impact on advancing diagnostic technologies. [ABSTRACT FROM AUTHOR]

Herein, we have prepared a 5,10,15,20-Tetrakis(4-hydroxyphenyl) porphyrin (P) which acts as a probe for selective and sensitive detection of Bi3+ ions. Probe P was obtained by reacting pyrrole with 4-hydroxyl benzaldehyde and characterized by NMR, IR, and ESI-MS. All photo-physical studies of P were tested in DMSO:H2O (8:2, v/v) media by spectrophotometry and spectrofluorometry respectively. The selectivity of P was tested with different metal ions in solution as well as in the solid phase, only Bi3+ showed red fluorescence quenching while with other metal ions, no such effect was observed. The Job's plot unveiled the 1:1 stoichiometric binding ratio of the probe with Bi3+ and anticipated association constant of 3.4 ×105 M-1, whereas the Stern-Volmer quenching constant was noticed to be 5.6 ×105 M-1. Probe P could detect Bi3+ down to 27 nM by spectrofluorometric. The binding mechanism of P with Bi3+ was well supported with NMR, mass, and DFT studies. Further, the P was applied for the quantitative determination of Bi3+ in various water samples and the biocompatibility of P was examined using neuro 2A (N2a) cells. Overall, probe P proves promising for the detection of Bi3+ in the semi-aqueous phase and it is the first report as a colorimetric and fluorogenic probe. [ABSTRACT FROM AUTHOR]

Two KCNA2 variants (p.H310Y and p.H310R) were discovered in paediatric patients with epilepsy and developmental delay. KCNA2 encodes KV1.2‐channel subunits, which regulate neuronal excitability. Both gain and loss of KV1.2 function cause epilepsy, precluding the prediction of variant effects; and while H310 is conserved throughout the KV‐channel superfamily, it is largely understudied. We investigated both variants in heterologously expressed, human KV1.2 channels by immunocytochemistry, electrophysiology and voltage‐clamp fluorometry. Despite affecting the same channel, at the same position, and being associated with severe neurological disease, the two variants had diametrically opposite effects on KV1.2 functional expression. The p.H310Y variant produced 'dual gain of function', increasing both cell‐surface trafficking and activity, delaying channel closure. We found that the latter is due to the formation of a hydrogen bond that stabilizes the active state of the voltage‐sensor domain. Additionally, H310Y abolished 'ball and chain' inactivation of KV1.2 by KVβ1 subunits, enhancing gain of function. In contrast, p.H310R caused 'dual loss of function', diminishing surface levels by multiple impediments to trafficking and inhibiting voltage‐dependent channel opening. We discuss the implications for KV‐channel biogenesis and function, an emergent hotspot for disease‐associated variants, and mechanisms of epileptogenesis. Key points: KCNA2 encodes the subunits of KV1.2 voltage‐activated, K+‐selective ion channels, which regulate electrical signalling in neurons. We characterize two KCNA2 variants from patients with developmental delay and epilepsy. Both variants affect position H310, highly conserved in KVchannels.The p.H310Y variant caused 'dual gain of function', increasing both KV1.2‐channel activity and the number of KV1.2 subunits on the cell surface.H310Y abolished 'ball and chain' (N‐type) inactivation of KV1.2 by KVβ1 subunits, enhancing the gain‐of‐function phenotype.The p.H310R variant caused 'dual loss of function', diminishing the presence of KV1.2 subunits on the cell surface and inhibiting voltage‐dependent channel opening.As H310Y stabilizes the voltage‐sensor active conformation and abolishes N‐type inactivation, it can serve as an investigative tool for functional and pharmacological studies. [ABSTRACT FROM AUTHOR]

Introduction: Pompe Disease is a type of lysosomal storage disease that is caused by a deficiency of the lysosomal alpha glucosidase. Pompe disease, as a multi-systemic disorder has a broad spectrum of clinical symptoms. The objective of this research was to validate and standardize the fluorometric method to detect alpha glucosidase activity, which can be used to identify patients with Pompe disease.Methods: This study was performed on 45 Pompe patients and 50 healthy control subjects. Dried blot spots were collected and subjected to an alpha glucosidase activity assay using the fluorometric method. The obtained fluorometric outcomes were then compared to those of the validated MS-MS method.Results: The control group showed a greater level of lysosomal alpha glucosidase activity compared to patients with Pompe disease (P value

Metal poisoning in humans can cause health hazards such as insomnia, mental retardation, nervous disorders and blindness. Most of the commonly used toxic metals are mercury, lead and cadmium. Among these toxic metals, mercury (Hg) has been declared to be highly toxic by the World Health Organization and Environmental Protection Agency. In this regard, the determination of Hg2+ in trace quantities plays an important role in the environment. This study investigates the preparation of embonic acid (EA) functionalized silver nanoparticles (AgNP) through eco-friendly approach for detection of mercuric ion in an aqueous environment. UV–Visible spectra analysis confirmed the stability of EA capped AgNP (EA-AgNP) over a period of 6 months. Stability and destabilization of silver nanoparticles in absence and presence of Hg2+ were confirmed by the zeta potential analysis with the value of − 28.5 mV and − 15 mV and High Resolution-Transmission Electronic Microscopy with increasing size of 15 nm to 130 nm respectively. The lower detection limit was found to be 0.5 nM and 50 fM Hg2+ using EA-AgNP by spectrophotometric and spectrofluorometric respectively and also highly selective to mercuric ion in the presence of a host of commonly interfering cations in aqueous system. [ABSTRACT FROM AUTHOR]

Novel BF2‐β‐diketonate complexes 1–4 bearing phenylboronic acid moieties and methoxyphenyl or dimethylaminophenyl groups were synthesized and their photophysical properties, pH response, and reactivity to D‐fructose were evaluated. Complexes 1 and 2 bearing a methoxyphenyl group luminesced with high intensity in a range of non‐polar to polar solvents, except in DMSO, whereas complexes 3 and 4 containing a dimethylaminophenyl group with twisted intramolecular charge transfer (TICT) properties showed a dramatic decrease in luminescence intensity with increasing solvent polarity. The luminescence intensity of complexes 1 and 2 decreased with increasing pH due to intermolecular vibration which associated with hydrogen bonding between the complexes and solvent water molecules, whereas it increased when they reacted with D‐fructose due to the suppression of the intermolecular vibration. In contrast, the luminescence intensity of complexes 3 and 4 increased with increasing pH and upon reaction with D‐fructose due to the loss of its TICT properties. The photophysical properties of complexes 1–4 and the TICT properties of complexes 3 and 4 were supported by density functional theory calculations. [ABSTRACT FROM AUTHOR]

The hydroxyl radicals (·OH) produced by the Fenton reaction of iron(II) and hydrogen peroxide (H2O2) can oxidize the colorless 3,3′,5,5′‐tetramethylbenzidine (TMB) to blue oxidized TMB (Ox‐TMB), resulting in a decrease in the fluorescence intensity of the reaction system and an increase in ultraviolet absorption. Ox‐TMB had a visible absorption peak at 625 nm and a fluorescence peak around 420 nm. When gallic acid (GA) was added to the system, Ox‐TMB was reduced to TMB, which made the color of the system disappear and the fluorescence recover. The linear ranges for determination of iron(II) were 0.5–10 μM (fluorometric) and 0.5–20 μM (colorimetric), and the detection limits were 0.25 μM (fluorometric) and 0.28 μM (colorimetric). The linear ranges for determination of GA were 0–80 μM (fluorometric) and 0–60 μM (colorimetric), and the detection limits were 0.31 μM (fluorometric) and 0.8 μM (colorimetric). The results of anti‐interference experiments shew that this dual‐mode assay had very good selectivity for the determination of iron(II) and GA. [ABSTRACT FROM AUTHOR]

Soft microfluidic systems that capture, store, and perform biomarker analysis of microliter volumes of sweat, in situ, as it emerges from the surface of the skin, represent an emerging class of wearable technology with powerful capabilities that complement those of traditional biophysical sensing devices. Recent work establishes applications in the real-time characterization of sweat dynamics and sweat chemistry in the context of sports performance and healthcare diagnostics. This paper presents a collection of advances in biochemical sensors and microfluidic designs that support multimodal operation in the monitoring of physiological signatures directly correlated to physical and mental stresses. These wireless, battery-free, skin-interfaced devices combine lateral flow immunoassays for cortisol, fluorometric assays for glucose and ascorbic acid (vitamin C), and digital tracking of skin galvanic responses. Systematic benchtop evaluations and field studies on human subjects highlight the key features of this platform for the continuous, noninvasive monitoring of biochemical and biophysical correlates of the stress state.

Key pointsKV 1.2 channels, encoded by the KCNA2 gene, regulate neuronal excitability by conducting K+ upon depolarization. A new KCNA2 missense variant was discovered in a patient with epilepsy, causing amino acid substitution F302L at helix S4, in the KV 1.2 voltage-sensing domain. Immunocytochemistry and flow cytometry showed that F302L does not impair KCNA2 subunit surface trafficking. Molecular dynamics simulations indicated that F302L alters the exposure of S4 residues to membrane lipids. Voltage clamp fluorometry revealed that the voltage-sensing domain of KV 1.2-F302L channels is more sensitive to depolarization. Accordingly, KV 1.2-F302L channels opened faster and at more negative potentials; however, they also exhibited enhanced inactivation: that is, F302L causes both gain- and loss-of-function effects. Coexpression of KCNA2-WT and -F302L did not fully rescue these effects. The proband's symptoms are more characteristic of patients with loss of KCNA2 function. Enhanced KV 1.2 inactivation could lead to increased synaptic release in excitatory neurons, steering neuronal circuits towards epilepsy.AbstractAn exome-based diagnostic panel in an infant with epilepsy revealed a previously unreported de novo missense variant in KCNA2, which encodes voltage-gated K+ channel KV 1.2. This variant causes substitution F302L, in helix S4 of the KV 1.2 voltage-sensing domain (VSD). F302L does not affect KCNA2 subunit membrane trafficking. However, it does alter channel functional properties, accelerating channel opening at more hyperpolarized membrane potentials, indicating gain of function. F302L also caused loss of KV 1.2 function via accelerated inactivation onset, decelerated recovery and shifted inactivation voltage dependence to more negative potentials. These effects, which are not fully rescued by coexpression of wild-type and mutant KCNA2 subunits, probably result from the enhancement of VSD function, as demonstrated by optically tracking VSD depolarization-evoked conformational rearrangements. In turn, molecular dynamics simulations suggest altered VSD exposure to membrane lipids. Compared to other encephalopathy patients with KCNA2 mutations, the proband exhibits mild neurological impairment, more characteristic of patients with KCNA2 loss of function. Based on this information, we propose a mechanism of epileptogenesis based on enhanced KV 1.2 inactivation leading to increased synaptic release preferentially in excitatory neurons, and hence the perturbation of the excitatory/inhibitory balance of neuronal circuits.

Measurement of ammonia in exhaled breath is of interest in medical and health sciences. The fluorometric determination of NH3 in exhaled breath condensate (EBC) was developed based on a digital image of a windowless falling drop (WFD) cell at the end of tube via a sensitive and selective reaction of NH3-OPA-sulfite in flow injection analysis system (FIA). A low-cost USB microscope as a facile instrumentation without expensive optical parts, an amplifier circuit, and a data acquisition unit, was used as a fluorescence light detector for the WFD cell via instant reporting temporal profile of digital value. A linear calibration graph of NH4+ in the range of 0.5–10 µmol L−1 can be obtained with a limit of detection of 0.5 µmol L−1 NH4+, and repeatability of 5.5% RSD (n = 11). The fluorescence (FL) signal obtained from the low-cost USB microscope was not different from that using the photodiode detector via a cross-shape flow cell. The proposed device was successfully applied for NH4+ determination in EBC and air absorbing solution samples with the recovery of 98–110%. The approach for instant reporting of a temporal profile of digital image value from a camera shows a potential to be applied for rapid chemical analysis in other microfluidics systems.

In present study, bovine serum albumin (BAS)-stabilized gold nanoclusters (AuNCs) combined with Cu2+ were used for rapid fluorimetric detection of glyphosate in water samples. The morphology and optical properties of synthesized BSA-AuNCs were characterized by TEM, UV–Vis spectroscopy and fluorescence techniques. Theoretically, Cu2+ would react with the tryptophan of BSA-AuNCs, thereby disrupting the surface structure of BSA-AuNCs and allowing aggregation of dispersed BSA-AuNCs, which showed the function to quenching the red fluorescence of BSA-AuNCs at 680 nm. However, in the presence of glyphosate in the system, there are strong chelation interactions between Cu2+ and glyphosate, so the fluorescence of BSA-AuNCs was restored and the recovered intensity is related to the glyphosate concentration. As a result, a linear graph between the fluorescence intensity with glyphosate concentration in the range of 0.06–12 μg/mL was plotted and used for quantification of glyphosate with a detection limit of 8 ng/mL. Satisfactory recoveries were obtained in the determination of spiked water samples, demonstrating that the prepared BSA-AuNCs can be used as a promising tool in the field of environment monitoring of glyphosate residue. [ABSTRACT FROM AUTHOR]

The fast, accurate detection of biomolecules, ranging from nucleic acids and small molecules to proteins and cellular secretions, plays an essential role in various biomedical applications. These include disease diagnostics and prognostics, environmental monitoring, public health, and food safety. Aptamer recognition (DNA or RNA) has gained extensive attention for biomolecular detection due to its high selectivity, affinity, reproducibility, and robustness. Concurrently, biosensing with nanoparticles has been widely used for its high carrier capacity, stability and feasibility of incorporating optical and catalytic activity, and enhanced diffusivity. Biosensors based on aptamers and nanoparticles utilize the combination of their advantages and have become a promising technology for detecting of a wide variety of biomolecules with high sensitivity, reliability, specificity, and detection speed. Via various sensing mechanisms, target biomolecules have been quantified in terms of optical (e.g., colorimetric and fluorometric), magnetic, and electrical signals. In this review, we summarize the recent advances in and compare different aptamer–nanoparticle-based biosensors by nanoparticle types and detection mechanisms. We also share our views on the highlights and challenges of the different nanoparticle-aptamer-based biosensors. [ABSTRACT FROM AUTHOR]

Too many ammonia emissions are released into the environment from cattle farming. These damage the environment and have an impact on animal and human health. Ammonia Emissions could be reduce by urease inhibitors. Before using the urease inhibitor suspension Atmowell® in cattle farming a risk assessment is required. This includes exposure data on the animal and human in the barn. As there is no method for exposure measurements yet the approach of fluorometry was taken. The fluorescent dye pyranine shall replace Atmowell® in later studies as a tracer. Before Atmowell® can be replaced, the interaction between Atmowell® and pyranine—according to the fluorescence and storage stability under the influence of ultraviolet light, has to be observed and excluded. Also, the spray and drift behavior must be examined in the wind tunnel with three different nozzles. The results show that Atmowell® has no effect on neither the fluorescence nor the degradation rate of a pyranine-solution. Furthermore, it is shown that a pyranine + Atmowell® mixture does not differ in drift behavior from a pure pyranine-solution. Because of these findings, an Atmowell®-solution can be substituted by a pyranine-solution without any effects on the results of an exposure measurement being expected. [ABSTRACT FROM AUTHOR]

This protocol describes a detailed fluorometric method for measuring peroxiredoxin (Prx) enzyme activity in vitro. Peroxide dissociation is the rate-limiting step in the Prx-controlled enzymatic reaction. To prevent interference by the catalase enzyme, we developed a peroxiredoxin assay that measures Prx activity using the substrate tert-Butyl hydroperoxide (t-BOOH). Prx enzyme activity is measured by incubating the enzymatic substrates 1,4-dithio-DL-threitol (DTT) and t-BOOH in a suitable buffer at 37 °C for 10 min in the presence of the desired volume of Prx enzyme. Next, the reagent N-(9-Acridinyl)maleimide (NAM) is used to stop the enzymatic reaction and form a fluorescent end product. Finally, Prx activity is measured by thiol fluorometry using a Box–Behnken design to optimize reaction conditions. This novel protocol was validated by evaluating Prx activity in matched samples against a reference assay. The correlation coefficient between our protocol and the reference assay was 0.9933, demonstrating its precision compared with existing methods. The NAM-Prx protocol instead uses t-BOOH as a substrate to measure Prx activity. Because catalase does not participate in the dissociation of t-BOOH, this approach does not require sodium azide. Furthermore, the method eliminates the need for concentrated acids to terminate the Prx enzymatic reaction since the NAM reagent can inhibit the enzymatic reaction regulated by the Prx enzyme. [ABSTRACT FROM AUTHOR]

Seasonally slowly melting mountain snowfields are populated by extremophilic microalgae. In alpine habitats, high‐light sensitive, green phytoflagellates are usually observed in subsurface layers deeper in the snowpack under dim conditions, while robust orange to reddish cyst stages can be seen exposed on the surface. In this study, uncommon surface green snow was investigated in the High Tatra Mountains (Slovakia). The monospecific community found in the green surface bloom consisted of vegetative Chloromonas cells (Volvocales, Chlorophyta). Molecular data demonstrated that the field sample and the strain isolated and established from the bloom were conspecific, and they represent a new species, Chloromonas kaweckae sp. nov., which is described based on the morphology of the vegetative cells and asexual reproduction and on molecular analyses of the strain. Cells of C. kaweckae accumulated approximately 50% polyunsaturated fatty acids, which is advantageous at low temperatures. In addition, this new species performed active photosynthesis at temperatures close to the freezing point showed a light compensation point of 126 ± 22 μmol photons · m−2 · s−1 and some signs of photoinhibition at irradiances greater than 600 μmol photons · m−2 · s−1. These data indicate that the photosynthetic apparatus of C. kaweckae could be regarded as adapted to relatively high light intensities, otherwise unusual for most flagellate stages of snow algae. [ABSTRACT FROM AUTHOR]

PurposeWe previously identified an oxysterol, VP1-001 (also known as compound 29), that partially restores the transparency of lenses with cataracts. To understand the mechanism of VP1-001, we tested the ability of its enantiomer, ent-VP1-001, to bind and stabilize αB-crystallin (cryAB) in vitro and to produce a similar therapeutic effect in cryAB(R120G) mutant and aged wild-type mice with cataracts. VP1-001 and ent-VP1-001 have identical physicochemical properties. These experiments are designed to critically evaluate whether stereoselective binding to cryAB is required for activity.MethodsWe compared the binding of VP1-001 and ent-VP1-001 to cryAB using in silico docking, differential scanning fluorimetry (DSF), and microscale thermophoresis (MST). Compounds were delivered by six topical administrations to mouse eyes over 2 weeks, and the effects on cataracts and lens refractive measures in vivo were examined. Additionally, lens epithelial and fiber cell morphologies were assessed via transmission electron microscopy.ResultsDocking studies suggested greater binding of VP1-001 into a deep groove in the cryAB dimer compared with ent-VP1-001. Consistent with this prediction, DSF and MST experiments showed that VP1-001 bound cryAB, whereas ent-VP1-001 did not. Accordingly, topical treatment of lenses with ent-VP1-001 had no effect, whereas VP1-001 produced a statistically significant improvement in lens clarity and favorable changes in lens morphology.ConclusionsThe ability of VP1-001 to bind native cryAB dimers is important for its ability to reverse lens opacity in mouse models of cataracts.

Pathogenic bacteria pose a serious threat to public health, and the rapid and cost-effective detection of such bacteria remains a major challenge. Herein, we present a DNAzyme-based fluorescent paper sensor for Klebsiella pneumoniae. The DNAzyme was generated by an in vitro selection technique to cleave a fluorogenic DNA-RNA chimeric substrate in the presence of K. pneumoniae. The DNAzyme was printed on a paper substrate in a 96-well format to serve as mix-and-read fluorescent assay that exhibits a limit of detection (LOD) 105  CFUs mL-1 . Evaluated with 20 strains of clinical bacterial isolates, the DNAzyme produced the desired fluorescence signal with the samples of K. pneumoniae, regardless of their source or drug resistance. The assay is simple to use, rapid, inexpensive, and avoids the complex procedures of sample preparation and equipment. We believe that this DNAzyme-based fluorescent assay has potential for practical applications to identify K. pneumoniae.

Agarose gel electrophoresis is a relatively easy to use method, commonly applied to evaluate PCR reaction success. Intercalating agents or dyes are used to visualize the amplified fragments. However, it is uncertain to what extent the brightness of bands is informative about the concentration of the amplicons. To more closely examine the suitability of agarose gel electrophoresis to assess PCR product yield, we quantified the brightness of bands on a gel and compared these data with the results from spectrophotometry, fluorometry and qPCR. Evaluation of the results suggests that assessment of the relative quantity of amplicons by band brightness is precise enough even for post-PCR analysis steps requiring PCR product concentrations within a certain range to function properly.

In this article; the bimetal metal-organic framework Zn/Mg (Zn/Mg MOF) is synthesized.Then Zn/Mg MOF bimetal was combined with Fe3O4 and Fe3O4@SiO2, and composites of Fe3O4@ SiO2/MOF/Dextrin, Fe3O4@SiO2/MOF, Fe3O4@MOF/Dextrin and Fe3O4@MOF made. The peroxidase-like activity of these compounds was investigated and compared by calorimetric Resazurin (Rz) and O-phenylenediamine (OPD); (Rz-H2O2, OPD-H2O2) and fluorimetric Rz and terephtalic acid (TA); (Rz-H2O2, TA-H2O2). The Fe3O4@ MOF/Dextrin composite has the highest peroxidase-like activity. The effect factors (amount of pH (6), the values of TA (1.37 mM), H2O2 (0.025 mM), reaction time (8.15 min), and amount of Composite (116.67 mg)) to increase the catalytic activity of Fe3O4@ MOF/Dextrin measured by chemometrics method. The most suitable linear range of the calibration curve by the TA-H2O2 -Composite fluorimetric method is 1–600 μg L−1, and the detection limit is 2.27 μg L−1. The relative standard deviation (RSD%) for measuring concentration atropine 1 μg L−1 (n = 6) is 1.18%. Finally, from this system for measuring atropine extracted by the Liquid-liquid extraction (LLE) method in two types of plants, D. Innoxia north and west and D. stramonium north and west of Iran (118.25 μg L−1, 79.80 μg L−1) and (18.477 μg L−1, 9.27 μg L−1) used, respectively.

Remdesivir is a prodrug that works by imitating the nucleoside adenosine. It is an antiviral medication that has been used to treat the coronavirus. Given the severity of the condition, the medicine is critical to the therapy and accompanying research. Remdesivir is extremely important as a medicinal dose form. Remdesivir has been used with other medications in the following combinations: Remdesivir and Dexamethasone, Remdesivir and Tocilizumab, and Remdesivir and Ivermectin. To aid future study, the paper also gives a simultaneous and comparative review of the analytical investigations published in the literature for a pharmacological estimate. The extensive literature searches turned up a plethora of study publications. A complete explanation of common, hyphenated, and unique ways of analysis is given. Remdesivir analytical quantification is provided using HPLC, LCMS, UV-Visible spectroscopy, and fluorescence. [ABSTRACT FROM AUTHOR]

Background: Cell-free DNA analysis (cfDNA) holds promise for residual disease or tumor burden quantification in colorectal cancer, with reduced costs and diagnostic equipment compared to gold standard-specific tumor DNA (ctDNA) analysis. Methods: This prospective case–control study included 46 colorectal cancer patients and healthy controls to perform cfDNA quantification by fluorometry using Quantus Fluorometer (Promega, Madison, WI) and using cell-free DNA ScreenTape assay (Agilent) and 4200 TapeStation instrument (Agilent Technologies, Inc., Santa Clara, CA, USA). cfDNA quantification results were correlated with stage, clinical and histopathological features. Results: 33 localized (8 stage I, 12 stage II, and 13 stage III) and 13 advanced colorectal cancer patients were included. No differences in cfDNA quantification by fluorometry were demonstrated depending on stage or histopathological features in localized disease patients. Differences in cfDNA quantification by fluorometry could be demonstrated in patients with advanced disease depending on the presence of liver metastases and synchronous or metachronous metastatic disease. Differences in cfDNA quantification by fluorometry could be demonstrated between advanced colorectal cancer patients and both localized disease patients and healthy controls. Secondary cfDNA analysis by electrophoresis, although showing more specificity to measure ctDNA in cfDNA values, could not improve the capacity to detect differences between analyzed a groups beyond previously achieved with fluorometry. Conclusion: This exploratory analysis of cfDNA based on fluorometry and electrophoresis methods showed promising results discriminating colorectal cancer and non-cancer patients, as well as different colorectal cancer stages and disease profiles. Further studies are needed to increase our knowledge and to help to overcome barriers to broader implementation and applications. [ABSTRACT FROM AUTHOR]

• We performed a comparative study on cyanobacterial risk monitoring methods. • Fluorometry and microscopy overestimated health risks associated to toxins. • qPCR and toxin analyses best captured health risks associated to toxins. • Toxin analyses provide the best public health risk assessment. • A two-tiered approach using multiple methods might be most cost-effective. Toxic cyanobacterial blooms impose a health risk to recreational users, and monitoring of cyanobacteria and associated toxins is required to assess this risk. Traditionally, monitoring for risk assessment is based on cyanobacterial biomass, which assumes that all cyanobacteria potentially produce toxins. While these methods may be cost effective, relatively fast, and more widely accessible, they often lead to an overestimation of the health risk induced by cyanotoxins. Monitoring methods that more directly target toxins, or toxin producing genes, may provide a better risk assessment, yet these methods may be more costly, usually take longer, or are not widely accessible. In this study, we compared six monitoring methods (fluorometry, microscopy, qPCR of 16S and mcyE , ELISA assays, and LC-MS/MS), of which the last three focussed on the most abundant cyanotoxin microcystins, across 11 lakes in the Netherlands during the bathing water season (May-October) of 2019. Results of all monitoring methods significantly correlated with LC-MS/MS obtained microcystin levels (the assumed 'golden standard'), with stronger correlations for methods targeting microcystins (ELISA) and microcystin genes (mcyE). The estimated risk levels differed substantially between methods, with 78 % and 56 % of alert level exceedances in the total number of collected samples for fluorometry and microscopy-based methods, respectively, while this was only 16 % and 6 % when the risk assessment was based on ELISA and LC-MS/MS obtained toxin concentrations, respectively. Integrating our results with earlier findings confirmed a strong association between microcystin concentration and the biovolume of potential microcystin-producing genera. Moreover, using an extended database consisting of 4265 observations from 461 locations across the Netherlands in the bathing water seasons of 2015 – 2019, we showed a strong association between fluorescence and the biovolume of potentially toxin-producing genera. Our results indicate that a two-tiered approach may be an effective risk assessment strategy, with first a biomass-based method (fluorometry, biovolume) until the first alert level is exceeded, after which the risk level can be confirmed or adjusted based on follow-up toxin or toxin gene analyses. [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H2S) is a colorless, foul smelling, toxic substance that can be found in water bodies and waste waters, especially in occupational susceptible environments, and can lead to harmful effects in humans at higher concentrations. An H2S monitoring probe NNAP is synthesized, which displays pH-dependent electrochemical, colorimetric, and fluorescence responses. NNAP functions as a fluorometric sensor at pH 7.4, with a limit of detection (LOD) of 0.70 mM, and as a colorimetric sensor at pH 12, where visible color changes are discernible to the naked eye, with an LOD of 4.28 mM. Additionally, it demonstrates utility in electrochemical sensing at pH 2, with a LOD of 2.5 mM. Furthermore, NNAP-coated paper strips have been successfully utilized for real-time H2S monitoring applications.Graphical Abstract: Hydrogen sulfide (H2S) is a colorless, foul smelling, toxic substance that can be found in water bodies and waste waters, especially in occupational susceptible environments, and can lead to harmful effects in humans at higher concentrations. An H2S monitoring probe NNAP is synthesized, which displays pH-dependent electrochemical, colorimetric, and fluorescence responses. NNAP functions as a fluorometric sensor at pH 7.4, with a limit of detection (LOD) of 0.70 mM, and as a colorimetric sensor at pH 12, where visible color changes are discernible to the naked eye, with an LOD of 4.28 mM. Additionally, it demonstrates utility in electrochemical sensing at pH 2, with a LOD of 2.5 mM. Furthermore, NNAP-coated paper strips have been successfully utilized for real-time H2S monitoring applications. [ABSTRACT FROM AUTHOR]

Endogenous circadian clocks control 24-h physiological and behavioral rhythms in mammals. Here, we report a real-time in vivo fluorescence recording system that enables long-term monitoring of circadian rhythms in the brains of freely moving mice. With a designed reporter of circadian clock gene expression, we tracked robust Cry1 transcription reporter rhythms in the suprachiasmatic nucleus (SCN) of WT, Cry1-/- , and Cry2-/- mice in LD (12 h light, 12 h dark) and DD (constant darkness) conditions and verified that signals remained stable for over 6 mo. Further, we recorded Cry1 transcriptional rhythms in the subparaventricular zone (SPZ) and hippocampal CA1/2 regions of WT mice housed under LD and DD conditions. By using a Cre-loxP system, we recorded Per2 and Cry1 transcription rhythms specifically in vasoactive intestinal peptide (VIP) neurons of the SCN. Finally, we demonstrated the dynamics of Per2 and Cry1 transcriptional rhythms in SCN VIP neurons following an 8-h phase advance in the light/dark cycle.

IntroductionVentilation system filters process recirculated indoor air along with outdoor air. This function inspires the idea of using the filter as an indoor bioaerosol sampler. While promising, there remains a need to investigate several factors that could limit the accuracy of such a sampling approach. Among the important factors are the dynamics of microbial assemblages on filter surfaces over time and the differential influence of outdoor versus recirculated indoor air.MethodsThis study collected ventilation system filter samples from an air handling unit on a regular schedule over a 21-week period and analyzed the accumulation patterns of biological particles on the filter both quantitatively (using fluorometry and qPCR) and in terms of microbial diversity (using 16S rDNA and ITS sequencing).ResultsThe quantitative result showed that total and bacterial DNA accumulated monotonically, rising to 41 ng/cm2 for total DNA and to 2.8 ng/cm2 for bacterial DNA over the 21-week period. The accumulation rate of bacterial DNA correlated with indoor occupancy level. Fungal DNA first rose to 4.0 ng/cm2 before showing a dip to 1.4 ng/cm2 between weeks 6 and 10. The dip indicated a possible artifact of this sampling approach for quantitative analysis as DNA may not be conserved on the filter over the months-long service period. The sequencing results indicate major contributions from outdoor air for fungi and from recirculated indoor air for bacteria. Despite the quantitative changes, the community structure of the microbial assemblages was stable throughout the 21-week sampling period, highlighting the robustness of this sampling method for microbial profiling.ConclusionThis study supports the use of ventilation system filters as indoor bioaerosol samplers, but with caveats: 1) an outdoor reference is required to properly understand the contribution of outdoor bioaerosols; and 2) there is a need to better understand the persistence and durability of the targeted organisms on ventilation system filters.

Abstract Probing the onset of micellization, or determining the critical micelle concentration (CMC), is of crucial importance while remains to be challenged by growing demand for extraordinary sensitivity and accuracy. Although fluorometry has attracted wide attention owing to its superiority in simplicity and sensitivity over other methods, the presence and fluctuation of background fluorescence of conventional fluorescent probes undermine the accuracy of CMC determination. Herein, a series of novel fluorescent probes without background fluorescence at a concentration below CMC owing to absolute aggregation‐caused quenching (aACQ) are utilized for sensitive and accurate measurement of CMC. The aACQ probes aggregate spontaneously and instantly in an aqueous environment owing to molecular π–π stacking with fluorescence quenching absolutely. Therefore, the absence of background fluorescence at a concentration below CMC clears relevant interference associated with conventional fluorophores. In this study, the new method is applied for versatile surfactants with CMCs ranging from nanomolar to millimolar concentrations, especially copolymers with ultralow CMC. The higher sensitivity and accuracy are highlighted by comparison with conventional probes.

In the human sodium glucose cotransporter (hSGLT1) cycle, the protein undergoes conformational changes where the sugar-binding site alternatively faces the external and internal surfaces. Functional site-directed fluorometry was used to probe the conformational changes at the sugar-binding site. Residues (Y290, T287, H83, and N78) were mutated to cysteines. The mutants were expressed in Xenopus laevis oocytes and tagged with environmentally sensitive fluorescent rhodamines [e.g., tetramethylrhodamine (TMR)-thiols]. The fluorescence intensity was recorded as the mutants were driven into different conformations using voltage jumps. Sugar binding and transport by the fluorophore-tagged mutants were blocked, but Na+ binding and the voltage-dependent conformational transitions were unaffected. Structural models indicated that external Na+ binding opened a large aqueous vestibule (600 Å3) leading to the sugar-binding site. The fluorescence of TMR covalently linked to Y290C, T287C, and H83C decreased as the mutant proteins were driven from the inward to the outward open Na+-bound conformation. The time courses of fluorescence changes (milliseconds) were close to the SGLT1 capacitive charge movements. The quench in rhodamine fluorescence indicated that the environment of the chromophores became more polar with opening of the external gates as the protein transitioned from the inward to outward facing state. Structural analyses showed an increase in polar side chains and a decrease in hydrophobic side chains lining the vestibule, and this was reflected in solvation of the chromophore. The results demonstrate the opening and closing of external gates in real time, with the accompanying changes of polarity of the sugar vestibule.

Pesticides in environmental samples pose significant risks to ecosystems and human health since they require precise and efficient detection methods. Imidacloprid (IMI), a widely used neonicotinoid insecticide, exemplifies these hazards due to its potential toxicity. This study addresses the urgent need for improved monitoring of such contaminants by introducing a novel fluorometric method for detecting IMI using nitrogen-doped graphite carbon dots (N-GCDs). The sensor operates by quenching fluorescence through the interaction of Cu 2+ ions with N-GCDs. Subsequently, IMI binds to the imidazole group, chelates with Cu 2+ , and restores the fluorescence of N-GCDs. This alternating fluorescence behavior allows for the accurate identification of both Cu 2+ and IMI. The sensor exhibits linear detection ranges of 20-100 nM for Cu 2+ and 10-140 μg/L for IMI, with detection limits of 18 nM and 1.2 μg/L, respectively. The high sensitivity of this sensor enables the detection of real-world samples, which underscores its potential for practical use in environmental monitoring and agricultural safety., (© 2024 John Wiley & Sons Ltd.)

This study presented a nanoparticle-enhanced aptamer-recognizing homogeneous detection system combined with a portable instrument (NASPI) to quantify lipoarabinomannan (LAM). This system leveraged the high binding affinity of aptamers, the high sensitivity of nanoparticle cascade amplification, and the stabilization effect of dual stabilizers (fructose and histone), and used probe-Cu 2+ to achieve LAM detection at concentrations ranging from 10 ag mL -1 to 100 fg mL -1 , with a limit of detection of 3 ag mL -1 using a fluorometer. It can also be detected using an independently developed handheld fluorometer or the red-green-blue (RGB) camera of a smartphone, with a minimum detection concentration of 10 ag mL -1 . We validated the clinical utility of the biosensor by testing the LAM in the urine of patients. Forty urine samples were tested, with positive LAM results in the urine of 18/20 tuberculosis (TB) cases and negative results in the urine of 6/10 latent tuberculosis infection cases and 10/10 non-TB cases. The assay results revealed a 100% specificity and a 90% sensitivity, with an area under the curve of 0.9. We believe that the NASPI biosensor can be a promising clinical tool with great potential to convert LAM into clinical indicators for TB patients.

In this study, an environmentally sustainable fluorimetric method for determination of Vonoprazan fumarate (VON) in dosage forms using nanoprobes consisting of nitrogen and sulfur co-doped carbon quantum dots (N, S-CQDs). The N, S-CQDs were prepared through a microwave-assisted method in 30 s. The resulting N, S-CQDs were characterized using transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS). They exhibit fluorescence emission at 460 nm after excitation at 385 nm with a high quantum yield (60%). The analytical approach for VON determination relies on the quenching effect exerted by VON on the native fluorescence intensity of CQDs. The quenching mechanism was investigated using Stern-Volmer plots. The proposed method demonstrates linearity across a concentration range 10-80 μM (4.6-36.8 μg/mL) with corresponding limits of detection and quantitation calculated as 2.17 μM (0.99 μg/mL) and 6.58 μM (3.02 μg/mL), respectively. The method has been effectively utilized for the determination of VON in the pharmaceutical samples. Statistical comparison with reported RP-HPLC has been performed to verify the accuracy and precision of the developed method. The environmental sustainability of the developed method has been thoroughly examined through various greenness metrics., (© 2024 John Wiley & Sons Ltd.)

Olopatadine (OLP) is widely utilized as an effective antihistaminic drug for alleviating ocular itching associated with allergic conjunctivitis. With its frequent usage in pharmacies, there arises a pressing need for a cost-effective, easily implementable, environmentally sustainable detection method with high sensitivity. This study presents a novel signal-on fluorimetric method for detecting OLP in both its pure form and aqueous humor. The proposed approach depends on enhancing the weak intrinsic fluorescence emission of OLP, achieving a remarkable increase of up to 680% compared to its intrinsic fluorescence. This enhancement is achieved by forming micelles around protonated OLP using an acetate buffer (pH 3.6) and incorporating a solution of sodium dodecyl sulfate (SDS) surfactant. A strong correlation (R = 0.9996) is observed between the concentration of OLP and fluorescence intensities ranging from 1.0 to 100.0 ng mL -1 with a limit of detection of 0.22 ng mL -1 . This described method is successfully employed for quantifying OLP in both its powder form and pharmaceutical eye drops. Furthermore, it demonstrates robust performance in determining OLP in artificial aqueous humor with a percentage recovery of 99.05 ± 1.51, with minimal interference from matrix interferents. Moreover, the greenness of the described method was evaluated., (© 2024 John Wiley & Sons Ltd.)

To maintain a green and sustainable environment for human beings, rapid detection of potentially toxic heavy metals like mercury (Hg(II)) has attracted great attention. Recently, sensors have been designed which can selectively detect Hg(II) over other common available cations and give a naked eye or fluorometric response. In the last two decades, the trend is shifting from bulky organic chemosensors toward nanoparticles due to their rapid response, low cost, eco-friendly and easy synthesis. In this review, promising nanoparticles-based sensors for Hg(II) detection are discussed. The nano-sensors are functionalized with nucleotide or other suitable materials which coordinate with Hg(II) ions and give clear color or fluorescence change. The operational mechanisms are discussed focusing on its four basic types. The nanoparticles-based sensors are even able to detect Hg in three different oxidation states (Hg(II), Hg(I) and Hg(0)). Recently, the trend has been shifted from ordinary nanoparticles to magnetic nanoparticles to simultaneously detect and remove Hg(II) ions from environmental samples. Furthermore, the nano-sensors for Hg(II) are compared with each other and with the reported organic chemosensors.

Chlorophyll-a is an essential climate variable. Chlorophyll-a in situ measurements are usually used for the validation of satellite images. Previous intercomparisons have shown that there can be substantial differences between in situ laboratories. In order to shed light on these differences, we arranged international chlorophyll-a intercomparisons with eight participating laboratories during 1–2 July 2021. We performed two dedicated transects through Bråviken bay (NW Baltic proper) and sampled four stations in each transect along a chlorophyll-a gradient. We took three surface replicates per laboratory and per station, i.e., 24 samples per laboratory. The samples were filtered through Whatman GF/F filters, and filters were frozen in liquid nitrogen and distributed in dry ice to all laboratories together with chlorophyll-a standards. The results between labs compared quite well. The mean normalized bias (MNB) of the standard measurements ranged between −23% and +19% for all laboratories and −7% to +19% for the Baltic Sea laboratories compared to high-performance liquid chromatography. The MNB of the two Bråviken transects ranged between −23 and +17% for all laboratories (compared to the median of all spectrophotometric and fluorometric measurements) and between −2 and +17% for the Baltic Sea laboratories. On average, the chlorophyll-a concentrations measured by the fluorometric method were about 13% higher than those measured by spectrophotometry, and fluorometry samples tended to have more scatter. The largest uncertainties seem to be caused by variable storage and extraction methods and are not fully captured in this intercomparison. This is demonstrated by analyzing historical comparisons revealing very large uncertainties (root mean square difference (RMSD) up to 109% and bias up to 68%), possibly due to too low filtration volumes and due to different extraction and storage methods. Our recommendation is to flash-freeze samples in liquid nitrogen and store them at −80°C. After storage, they should be extracted and measured at room temperature within 6–24 h. Our results also indicate that ethanol is much more efficient in extracting Chl-a than acetone. Last but not least, we would like to point out that the uncertainties in measuring chlorophyll-a by satellite are now within the range of in situ data, as shown here by comparing the in situ results from this study with published remote sensing results from the literature.

The fast, accurate detection of biomolecules, ranging from nucleic acids and small molecules to proteins and cellular secretions, plays an essential role in various biomedical applications. These include disease diagnostics and prognostics, environmental monitoring, public health, and food safety. Aptamer recognition (DNA or RNA) has gained extensive attention for biomolecular detection due to its high selectivity, affinity, reproducibility, and robustness. Concurrently, biosensing with nanoparticles has been widely used for its high carrier capacity, stability and feasibility of incorporating optical and catalytic activity, and enhanced diffusivity. Biosensors based on aptamers and nanoparticles utilize the combination of their advantages and have become a promising technology for detecting of a wide variety of biomolecules with high sensitivity, reliability, specificity, and detection speed. Via various sensing mechanisms, target biomolecules have been quantified in terms of optical (e.g., colorimetric and fluorometric), magnetic, and electrical signals. In this review, we summarize the recent advances in and compare different aptamer–nanoparticle-based biosensors by nanoparticle types and detection mechanisms. We also share our views on the highlights and challenges of the different nanoparticle-aptamer-based biosensors.

Protein l-isoaspartyl methyltransferase (PIMT/PCMT1), a product of the human pcmt1 gene, catalyzes repair of abnormal l-isoaspartyl linkages in age-damaged proteins. Pcmt1 knock-out mice exhibit a profound neuropathology and die 30-60 days postnatal from an epileptic seizure. Here we express 15 reported variants of human PIMT and characterize them with regard to their enzymatic activity, thermal stability, and propensity to aggregation. One mutation, R36C, renders PIMT completely inactive, whereas two others, A7P and I58V, exhibit activity that is 80-100% higher than wild type. G175R is highly prone to aggregation and has greatly reduced activity. R17S and R17H show markedly enhanced sensitivity to thermal denaturation. Based on previous studies of moderate PIMT variation in humans and mice, we predict that heterozygosity for R36C, G175R, R17S, and R17H will prove detrimental to cognitive function and successful aging, whereas homozygosity (if it ever occurs) will lead to severe neurological problems in the young.

Low high-density lipoprotein cholesterol (HDL-C) levels are one of the most powerful independent negative predictors of atherosclerotic cardiovascular disease (CVD). The structure and function of HDL rather than HDL-C may more accurately predict atherosclerosis. Several HDL protein and lipid compositional changes that impair HDL function occur in inflammatory states such as atherosclerosis. HDL function is usually determined by cell based assays such as cholesterol efflux assay but these assays have numerous drawbacks lack of standardization. Cell-free assays may give more robust measures of HDL function compared to cell-based assays. HDL oxidation impairs HDL function. HDL has a major role in lipid peroxide transport and high amount of lipid peroxides is related to abnormal HDL function. Lipid-probe interactions should be considered when interpreting the results of non-enzymatic fluorescence assays for measuring the lipid oxidative state. This motivated us to develop a cell-free biochemical enzymatic method to assess HDL lipid peroxide content (HDLox) that contributes to HDL dysfunction. This method is based on the enzyme horseradish peroxidase (HRP) and the fluorochrome Amplex Red that can quantify (without cholesterol oxidase) the lipid peroxide content per mg of HDL-C. Here a protocol is describedfor determination of HDL-lipid peroxidation using the fluorochrome reagent. Assay variability can be reduced by strict standardization of experimental conditions. Higher HDLox values are associated with reduced HDL antioxidant function. The readout of this assay is associated with readouts of validated cell-based assays, surrogate measures of cardiovascular disease, systemic inflammation, immune dysfunction, and associated cardiovascular and metabolic risk phenotypes. This technical approach is a robust method to assess HDL function in human disease where systemic inflammation, oxidative stress and oxidized lipids have a key role (such as atherosclerosis).

[Display omitted] • A novel NIR fluorescent probe was designed for GSH detection. • The probe is thioctic acid-modified Ag/Au NCs (ThA@ Ag/Au NCs). • In presence of Fe3+, ThA@ Ag/Au NCs was aggregated, decreasing fluorescence emission. • Addition of GSH, it chelated Fe3+ from the surface of NCs, recovering the fluorescence emission. • The fluorescent probe was used to detect GSH in different biological samples. Accurate determination of glutathione (GSH) levels in biological fluids and food supplements is paramount. To address this need, we developed a near-infrared (NIR) fluorescent probe with high sensitivity and selectivity for GSH detection. This probe comprises thioctic acid-functionalized silver and gold nanoclusters (ThA@Ag/Au NCs), emitting red light at 720 nm upon excitation at 310 nm. In the presence of Fe3+ ions, which form complexes with ThA, the fluorescence of ThA@Ag/Au NCs is quenched due to aggregation. However, upon addition of GSH, a potent chelator for Fe3+, the fluorescence of the probe is restored, leading to enhanced emission. Under optimized conditions, the fluorescence response (F/F°) exhibits a linear relationship with increasing GSH concentration within the range of 0.03–95 µM, achieving a limit of detection (LOD) of 9.0 nM. This designed scheme demonstrates excellent selectivity, sensitivity, and accuracy for GSH detection. Furthermore, the NIR fluorescent probe effectively estimated GSH levels in various matrices, including capsules, serum, urine, and saliva samples, yielding recoveries % ranging from 97.2 % to 103.2 %, with RSD % below 4.34 %. Due to its NIR properties, this fluorescent probe holds promise for diagnosing various diseases and potential in vivo applications. [ABSTRACT FROM AUTHOR]

The measurement of phenylalanine in biological fluids for the diagnosis of phenylketonuria (PKU) in newborns and the monitoring/therapeutic drug monitoring of individuals with PKU are especially important. Owing to the importance of PKU monitoring in clinical medicine, a new fluorometric method was developed for the determination of L-phenylalanine in serum samples. This method is based on the relationship between phenylalanine ammonia-lyase (PAL) and o-phthalaldehyde (OPA). PAL catalyzes the conversion of phenylalanine to ammonia and trans-cinnamic acid. The formed ammonia reacts with OPA in the presence of sodium sulfite, giving a fluorescent product. The presence of sulfide in an alkaline environment prevents OPA from reacting with other amino acids while allowing it to react only with ammonia. Method characterization and optimization studies, such as the effects of pH, temperature, and interferents, were carried out. The amount of L-phenylalanine in a human serum sample was successfully determined by using the fluorescence emission intensity of the fluorescent product formed as a result of the reaction between OPA and ammonia. The linear range of the method is between 10 μM and 10 mM. The obtained result showed good agreement with the results of the biochemistry analysis laboratory. Recoveries of 95.41% and 73.39% were obtained for phenylalanine and ammonia, respectively. This PAL–OPA–based fluorometric method for phenylalanine is practical, easy to operate, low cost, highly sensitive, and selective and can also be used for the simultaneous determination of ammonia in human serum samples. [ABSTRACT FROM AUTHOR]

Intrinsic fluorescence analysis is a sensitive technique to gain insight about aptamer-small molecule interactions. Employing fluorescence properties of the ligand, the binding affinities of the aptamer-ligand complex can be quantified in the nM to mM range with great accuracy and precision. Here, we present a detection method for aptamer-ligand binding analysis that is based on the inherent fluorescence of the ligand. Further, we discuss how to optimize and resolve some common experimental challenges. [ABSTRACT FROM AUTHOR]