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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.

Spray penetration into citrus canopies is critical for adequate coverage and deposition to ensure effective pest control. However, mismatch of air assistance to target canopy characteristics can lead to unintended spraying losses through overpenetration. To evaluate the effect of air assistance on on-target deposition, two sprayers (surrogates for airflow rates) were used to apply a fluorescent tracer dye solution @ a target concentration of 300 ppm to 16 medium-foliage-density tree blocks in a commercial mandarin orchard. The complete factorial experiment in three replications, also designed for validating a model-based spray decision support tool, comprised two forward travel speeds (1.6 and 4.8 km/h), two disc-core nozzles (TeeJet® D3-25 and D6-45), and either one or two nozzle rows to obtain a wide range of application rates (496 to 9719 L/ha). Dye deposition significantly decreased with canopy depth (p =< 0.001) by nearly seven times across the 3.4 m wide canopies but was not significant over 1.2 to 2.2 m sampling height (p = 0.867). Deposition obtained with the low-airflow-rate sprayer was significantly greater (p =< 0.001) than that obtained with the high-airflow-rate sprayer over the dose range likely due to too much air pushing out spray droplets. This study underscores the importance of matching the air assistance of orchard sprayers to the target canopy. [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 non-invasive and efficient alternative for early cancer diagnosis showcasing its potential impact on advancing diagnostic technologies.

The escalating demand for versatile and precise diagnostic tools across various biomedical applications has led to the development of an advanced assay utilizing a fluorescent metal-organic framework (MOF). Our novel approach centers on the synthesis of a controlled-explosive NH2-MIL-53(Fe) MOF, composed of trivalent iron ions and 2-aminoterephthalic acid. This framework possesses the unique capability to produce explosively enhanced fluorescence upon environmental triggers such as phosphate conditions, where it disintegrates to release intensely fluorescent molecules. The surface of NH2-MIL-53(Fe) MOF was further functionalized with poly(acrylic acid-co-maleic acid) polymers (PAAcoMA) to form PAAcoMA@MIL-53(Fe) MOF, thereby enhancing its stability. The presence of phosphate ions is detected by the degree of fluorescence resulting from the unquenching of PAAcoMA@MIL-53(Fe) MOF. This system demonstrates not only high sensitivity but also a broad dynamic range, making it suitable for phosphate ion detection. This innovative technology holds promise for significant advancements in the field of phosphate indicator-based biosensing, with potential applications in the development of PCR kits, thereby supporting a wide range of diagnostic and therapeutic applications.

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]

Spray penetration into citrus canopies is critical for adequate coverage and deposition to ensure effective pest control. However, mismatch of air assistance to target canopy characteristics can lead to unintended spraying losses through overpenetration. To evaluate the effect of air assistance on on-target deposition, two sprayers (surrogates for airflow rates) were used to apply a fluorescent tracer dye solution @ a target concentration of 300 ppm to 16 medium-foliage-density tree blocks in a commercial mandarin orchard. The complete factorial experiment in three replications, also designed for validating a model-based spray decision support tool, comprised two forward travel speeds (1.6 and 4.8 km/h), two disc-core nozzles (TeeJet® D3-25 and D6-45), and either one or two nozzle rows to obtain a wide range of application rates (496 to 9719 L/ha). Dye deposition significantly decreased with canopy depth (p =< 0.001) by nearly seven times across the 3.4 m wide canopies but was not significant over 1.2 to 2.2 m sampling height (p = 0.867). Deposition obtained with the low-airflow-rate sprayer was significantly greater (p =< 0.001) than that obtained with the high-airflow-rate sprayer over the dose range likely due to too much air pushing out spray droplets. This study underscores the importance of matching the air assistance of orchard sprayers to the target canopy.

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

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.

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]

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.

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.

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.

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]

The autofluorescence of erythrocyte porphyrins has emerged as a potential method for multi-cancer early detection (MCED). With this method's dependence on research-grade spectrofluorometers, significant improvements in instrumentation are necessary to translate its potential into clinical practice, as with any promising medical technology. To fill this gap, in this paper, we present an automated ratio porphyrin analyzer for cancer screening (ARPA-CS), a low-cost, portable, and automated instrument for MCED via the ratio fluorometry of porphyrins. The ARPA-CS aims to facilitate cancer screening in an inexpensive, rapid, non-invasive, and reasonably accurate manner for use in primary clinics or at point of care. To accomplish this, the ARPA-CS uses an ultraviolet-excited optical apparatus for ratio fluorometry that features two photodetectors for detection at 590 and 630 nm. Additionally, it incorporates a synchronous detector for the precision measurement of signals based on the Walsh-ordered Walsh-Hadamard transform (WHT) w and circular shift. To estimate its single-photodetector capability, we established a linear calibration curve for the ARBA-CS exceeding four orders of magnitude with a linearity of up to 0.992 and a low detection limit of 0.296 µg/mL for riboflavin. The ARPA-CS also exhibited excellent repeatability (0.21%) and stability (0.60%). Moreover, the ratio fluorometry of three serially diluted standard solutions of riboflavin yielded a ratio of 0.4, which agrees with that expected based on the known emission spectra of riboflavin. Additionally, the ratio fluorometry of the porphyrin solution yielded a ratio of 49.82, which was ascribed to the predominant concentration of protoporphyrin IX in the brown eggshells, as confirmed in several studies. This study validates this instrument for the ratio fluorometry of porphyrins as a biomarker for MCED. Nevertheless, large and well-designed clinical trials are necessary to further elaborate more on this matter.

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.

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).

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.

Decaprenylphosphoryl-β-D-ribose-oxidase (DprE1), a subunit of the essential decaprenylphosphoribose-2'-epimerase, plays a crucial role in the synthesis of cell wall arabinan components in mycobacteria, including the pathogen responsible for tuberculosis, Mycobacterium tuberculosis . In this study, we designed, synthesised, and evaluated 15 (BOK-1-BOK-10 and BOP-1-BOP-5) potential inhibitors of DprE1 from a series of 1,2,3-triazole ligands using a validated DprE1 inhibition assay. Two compounds, BOK-2 and BOK-3, demonstrated significant inhibition with IC 50 values of 2.2 ± 0.1 and 3.0 ± 0.6 μM, respectively, whereas the standard drug (TCA-1) showed inhibition at 3.0 ± 0.2 μM. Through molecular modelling and dynamic simulations, we explored the structural relationships between selected 1,2,3-triazole compounds and DprE1, revealing key features for effective drug-target interactions. This study introduces a novel approach for designing ligands against DprE1, offering a potential therapeutic strategy for tuberculosis treatment.

Differential scanning fluorimetry (DSF) is used to assess protein stability, transition states, or the Kd values of various ligands, drug molecules, and antibodies. All fluorescent probes published to date either are incompatible with hydrophobic proteins/ligands, precluding analyses of transmembrane or membrane-associated proteins, or have excitation and detection wavelengths outside the range of real-time polymerase chain reaction (RT-PCR) machines, necessitating the use of dedicated devices. Here, we describe a thiol-reactive probe, BODIPY FL L-cystine (BFC), to overcome both of these shortcomings. The probe supports an inexpensive application of DSF measurements suitable for detection with standard RT-PCR machines in a hydrophilic or hydrophobic environment.

BackgroundLaboratory diagnosis of erythropoietic protoporphyria (EPP) requires a marked increase in total erythrocyte protoporphyrin (300-5000 μg/dL erythrocytes, reference interval

Cinchona ledgeriana Moens is an industrial plant producing secondary metabolite quinoline alkaloids. To maintain and moreover, to increase the quinoline production especially quinine, in vitro culture system through cell culture could be a potential alternative. If the use of elicitor in cell culture can increase the production of a secondary metabolite, the activity of the enzymes involved in the biosynthetic pathway of the secondary metabolite in question might be increasing. This study aimed to examine the activity of NADPH oxidoreductase in the elicitated cell culture of C. ledgeriana and to evaluate the correlation between the activity of this enzyme and the level of quinine production. The cell cultures of Cinchona were treated with abscisic acid (ABA) or paclobutrazol (PBZ), combined with sucrose, sorbitol, or mannitol in Wood Plant (WP) media, for 7 weeks on a shaker. The quinine concentration was determined using high-performance liquid chromatography (HPLC) and the enzyme activity was measured using fluorometry. The results showed that the highest enzyme activity was found in the P7M cells (PBZ 7 mg/L + mannitol 5.3 g/L + sucrose 20 g/L), followed by the A3S cells (ABA 3 mg/L + sorbitol 5.3 g/L + sucrose 20 g/L). These results correspond to their production level of the quinine alkaloids. The lowest enzyme activity was found in the cultures without elicitor. The increase of NADP(H) enzyme activity in the P7M and A3S treatments were 13.5 and 8.5%, respectively, compared to that in the control cells.

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation channels involved in fast synaptic transmission. Pharmacological inhibition of ASIC1a reduces neurotoxicity and stroke infarct volumes, with the cysteine knot toxin psalmotoxin-1 (PcTx1) being one of the most potent and selective inhibitors. PcTx1 binds at the subunit interface in the extracellular domain (ECD), but the mechanism and conformational consequences of the interaction, as well as the number of toxin molecules required for inhibition, remain unknown. Here, we use voltage-clamp fluorometry and subunit concatenation to decipher the mechanism and stoichiometry of PcTx1 inhibition of ASIC1a. Besides the known inhibitory binding mode, we propose PcTx1 to have at least two additional binding modes that are decoupled from the pore. One of these modes induces a long-lived ECD conformation that reduces the activity of an endogenous neuropeptide. This long-lived conformational state is proton-dependent and can be destabilized by a mutation that decreases PcTx1 sensitivity. Lastly, the use of concatemeric channel constructs reveals that disruption of a single PcTx1 binding site is sufficient to destabilize the toxin-induced conformation, while functional inhibition is not impaired until two or more binding sites are mutated. Together, our work provides insight into the mechanism of PcTx1 inhibition of ASICs and uncovers a prolonged conformational change with possible pharmacological implications.

In this study, we attempted to develop a new boron quantification reagent using β-diketones, because β-diketones often exhibit strong absorption and luminescence and react with boric acid. The luminescence intensity remarkably varied with the addition of boric acid to a solution of β-diketone containing 4-methoxyphenyl groups. However, luminescence intensity gradually decreased with time because of the tautomerization of the enol form of β-diketone to its keto form. Therefore, in this study, we synthesized novel β-diketone–BF2 compounds in which the β-diketone site was cross-linked with BF2 to avoid changes in the luminescence intensity owing to the keto-enol tautomerization. We introduced a catechol moiety as a reaction site with boron and examined their effectiveness in quantifying boron using absorption and emission spectroscopy. Furthermore, the synthesized β-diketone–BF2 compounds (1–9) that contained 4-dimethylaminophenyl (4-NMe2Ph) or 4-methoxyphenyl (4-MeOPh) group and catechol or salicylic acid moiety as the reaction site, were characterized using various analytical methods. No correlation was observed between the pKa values of β-diketone–BF2 compounds and the formation constants for the reactions with boric acid (log K1). Additionally, quantification limit estimation carried out using absorption and emission spectroscopy suggested that β-diketone–BF2 compounds 3 and 7, which contain 4-NMe2Ph group and catechol moiety, can be used to determine boric acid concentrations > ca. 2 × 10–4 M. This study demonstrated the usefulness of the β-diketone-BF2 compound as a colorimetric and fluorescence quantifying reagent for boric acid.

Macrozones are novel conjugates of azithromycin and thiosemicarbazones, which exhibit very good in vitro antibacterial activities against susceptible and some resistant bacterial strains thus showing a potential for further development. A combination of spectrometric (fluorimetry, STD and WaterLOGSY NMR) and molecular docking studies provided insights into atomic details of interactions between selected macrozones and biological receptors such as E. coli ribosome and bovine serum albumin. Fluorimetric measurements revealed binding constants in the micro-molar range while NMR experiments provided data on binding epitopes. It has been demonstrated that both STD and WaterLOGSY gave comparable and consistent results unveiling atoms in intimate contacts with biological receptors. Docking studies pointed towards main interactions between macrozones and E. coli ribosome which included specific π - π stacking and hydrogen bonding interactions with thiosemicarbazone part extending down the ribosome exit tunnel. The results of the docking experiments were in fine correlation with those obtained by NMR and fluorimetry. Our investigation pointed towards a two-site binding mechanism of interactions between macrozones and E. coli ribosome which is the most probable reason for their activity against azithromycin-resistant strains. Much better activity of macrozone-nickel coordinated compound against E. coli ribosome compared to other macrozones has been attributed to the higher polarity which enabled better bacterial membrane penetration and binding of the two thiosemicarbazone units thus additionally contributing to the overall binding energy. The knowledge gained in this study should play an important role in anti-infective macrolide design in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purification of recombinant proteins for biochemical assays and structural studies is time-consuming and presents inherent difficulties that depend on the optimization of protein stability. The use of dyes to monitor thermal denaturation of proteins with sensitive fluorescence detection enables rapid and inexpensive determination of protein stability using real-time PCR instruments. By screening a wide range of solution conditions and additives in a 96-well format, the thermal shift assay easily identifies conditions that significantly enhance the stability of recombinant proteins. The same approach can be used as an initial low-cost screen to discover new protein-ligand interactions by capitalizing on increases in protein stability that typically occur upon ligand binding. This unit presents a methodological workflow for small-scale, high-throughput thermal denaturation of recombinant proteins in the presence of SYPRO Orange dye.

A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The VHH (variable domain of heavy chain antibody) gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double-mutant gene. The Nb28-AP construct was transformed into Escherichia coli BL21(DE3)plysS, and soluble expression in bacteria was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. Both the Nb properties and AP enzymatic activity were validated by colorimetric and fluorometric analysis. The 50% inhibitory concentration and the detection limit of the dc-FEIA were 0.13 and 0.04 ng/mL, respectively, with a linear range of 0.06-0.43 ng/mL. This assay was compared with LC-MS/MS, and the results indicated the reliability of Nb-AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal.

Improved tools for providing specific intraoperative diagnoses could improve patient care. In neurosurgery, intraoperatively differentiating non-operative lesions such as CNS B-cell lymphoma from operative lesions can be challenging, often necessitating immunohistochemical (IHC) procedures which require up to 24-48 hours. Here, we evaluate the feasibility of generating rapid ex vivo specific labeling using a novel lymphoma-specific fluorescent switchable aptamer. Our B-cell lymphoma-specific switchable aptamer produced only low-level fluorescence in its unbound conformation and generated an 8-fold increase in fluorescence once bound to its target on CD20-positive lymphoma cells. The aptamer demonstrated strong binding to B-cell lymphoma cells within 15 minutes of incubation as observed by flow cytometry. We applied the switchable aptamer to ex vivo xenograft tissue harboring B-cell lymphoma and astrocytoma, and within one hour specific visual identification of lymphoma was routinely possible. In this proof-of-concept study in human cell culture and orthotopic xenografts, we conclude that a fluorescent switchable aptamer can provide rapid and specific labeling of B-cell lymphoma, and that developing aptamer-based labeling approaches could simplify tissue staining and drastically reduce time to histopathological diagnoses compared with IHC-based methods. We propose that switchable aptamers could enhance expeditious, accurate intraoperative decision-making.

Excitation-evoked Ca(2+) influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, muscle contraction, and gene expression. The voltage dependence and timing of Ca(2+) entry are thought to be functions of voltage-gated calcium (CaV) channels composed of a central pore regulated by four nonidentical voltage-sensing domains (VSDs I-IV). Currently, the individual voltage dependence and the contribution to pore opening of each VSD remain largely unknown. Using an optical approach (voltage-clamp fluorometry) to track the movement of the individual voltage sensors, we discovered that the four VSDs of CaV1.2 channels undergo voltage-evoked conformational rearrangements, each exhibiting distinct voltage- and time-dependent properties over a wide range of potentials and kinetics. The voltage dependence and fast kinetic components in the activation of VSDs II and III were compatible with the ionic current properties, suggesting that these voltage sensors are involved in CaV1.2 activation. This view is supported by an obligatory model, in which activation of VSDs II and III is necessary to open the pore. When these data were interpreted in view of an allosteric model, where pore opening is intrinsically independent but biased by VSD activation, VSDs II and III were each found to supply ∼50 meV (∼2 kT), amounting to ∼85% of the total energy, toward stabilizing the open state, with a smaller contribution from VSD I (∼16 meV). VSD IV did not appear to participate in channel opening.

Obesity is a global disease that has been escalating to epidemic proportions over the past years. A recent report from World Obesity Federation predicts that, in 2030, 1 billion people will be obese. Thus, it is mandatory to develop new therapeutic options that can manage and control obesity. One of the most promising research paths is the inhibition of pancreatic lipase (PL), responsible for the hydrolysis of 50 to 70% of total dietary triglycerides. Chalcones are the precursors of flavonoids, consisting of two benzene rings connected by a three-carbon α, β-unsaturated carbonyl structure. The goal of the present work was to evaluate the activity of seven chalcones with hydroxy (OH) and chloride (Cl) substituents, as potential inhibitors of PL. For this purpose, spectrophotometric and fluorometric microanalysis systems were used, based on the enzymatic metabolization of p-nitrophenyl butyrate and 4-methylumbeliferyl oleate, respectively. The obtained results showed that chalcones inhibit PL activity, and that the fluorometric method can reach higher inhibition rates with fewer compounds than the spectrophotometric method. These findings bring new insights into the structure design for the modulation of PL, but further studies are still needed to further explore these compounds as potential anti-obesity molecules.

Coral reefs are declining worldwide due to global changes in the marine environment. The increasing frequency of massive bleaching events in the tropics is highlighting the need to better understand the stages of coral physiological responses to extreme conditions. Moreover, like many other coastal regions, coral reef ecosystems are facing additional localized anthropogenic stressors such as nutrient loading, increased turbidity, and coastal development. Different strategies have been developed to measure the health status of a damaged reef, ranging from the resolution of individual polyps to the entire coral community, but techniques for measuring coral physiology in situ are not yet widely implemented. For instance, while there are many studies of the coral holobiont response in single or limited-number multiple stressor experiments, they provide only partial insights into metabolic performance under more complex and temporally and spatially variable natural conditions. Here, we discuss the current status of coral reefs and their global and local stressors in the context of experimental techniques that measure core processes in coral metabolism (respiration, photosynthesis, and biocalcification) in situ, and their role in indicating the health status of colonies and communities. We highlight the need to improve the capability of in situ studies in order to better understand the resilience and stress response of corals under multiple global and local scale stressors.

A monitor calibrator has been tested as a portable, available, and cost-effective tool for the detection of luminescence. Prospects of this device as an all-sufficient analog of an analytical fluorometer have been outlined. Its ability to determine luminescent compounds was demonstrated by the example of rhodamine 3B and fluorescein. The determination is based on simultaneous impulse irradiation of a fluorescent sample with the incorporated continuous-spectrum light emitter in the visible and near UV ranges and registration of the luminescence. The appropriate cuvettes for the analysis and the device sealing film were chosen. The lowest relative detection limit for the developed approach was $0.03~\mu $ mol L−1 for rhodamine 3B and $0.01~\mu $ mol L−1 for fluorescein; the lowest absolute detection limits were 0.01 and 0.002 nmol for rhodamine 3B and fluorescein, respectively. Using the proposed approach, it was possible to determine the luminescent compounds in tap water. [ABSTRACT FROM AUTHOR]

Coral reefs are declining worldwide due to global changes in the marine environment. The increasing frequency of massive bleaching events in the tropics is highlighting the need to better understand the stages of coral physiological responses to extreme conditions. Moreover, like many other coastal regions, coral reef ecosystems are facing additional localized anthropogenic stressors such as nutrient loading, increased turbidity, and coastal development. Different strategies have been developed to measure the health status of a damaged reef, ranging from the resolution of individual polyps to the entire coral community, but techniques for measuring coral physiology in situ are not yet widely implemented. For instance, while there are many studies of the coral holobiont response in single or limited-number multiple stressor experiments, they provide only partial insights into metabolic performance under more complex and temporally and spatially variable natural conditions. Here, we discuss the current status of coral reefs and their global and local stressors in the context of experimental techniques that measure core processes in coral metabolism (respiration, photosynthesis, and biocalcification) in situ , and their role in indicating the health status of colonies and communities. We highlight the need to improve the capability of in situ studies in order to better understand the resilience and stress response of corals under multiple global and local scale stressors. [ABSTRACT FROM AUTHOR]

PurposeTo investigate factors of ethnicity, sex, age, and diurnal variation on human corneal epithelial permeability.MethodsData of corneal epithelial permeability to sodium fluorescein (Pdc) were collected from 374 noncontact lens wearers at various times after awakening throughout the day. Mixed-effect models were developed to investigate the association between Pdc and factors of interest, including time awake (TA), age, sex, ethnicity, and interactions of these factors.ResultsTwo models evaluated the "Pdc recovery period" from awakening to 2 hours (Model 1) and the "Pdc plateau" period after TA of 2 hours (Model 2). In Model 1, Pdc declined significantly with length of awake time (P = 0.000), and showed higher Pdc with males (P = 0.098), although this sex difference was not observed after 2 hours (Model 2). Both models showed significantly higher Pdc in Asians than in non-Asians (P = 0.000) and increased Pdc with age (P = 0.048, P = 0.001).ConclusionsBaseline corneal epithelial barrier function increases after overnight sleep and varies significantly by ethnicity and age.

PurposeTo determine whether daily irrigation with sterile saline solution during silicone hydrogel (SiH) contact lens continuous wear (CW) can mitigate increases in corneal epithelial permeability (Pdc) and reduce the risk of adverse events.MethodsIn this study, 161 non-contact lens wearers were fit with SiH contact lenses and randomized to either a treatment (n = 81) or control (n = 80) group for 30-day CW. Subjects in the treatment group irrigated every morning and whenever dryness symptoms occurred; subjects in the control group did not. Mixed effects models were employed to assess the changes in Pdc, and survival analysis methods were employed to estimate the risk for adverse events.ResultsEpithelial permeability increased over 30-day CW with SiH lenses (P = 0.001). Risk of inflammatory (odds ratio [OR] = 1.94) and lens-induced (OR = 1.90) adverse events increased with irrigation; these events also occurred sooner, on average, in the irrigation group. Although the overall risk for Asians was higher than for non-Asians, irrigation had no significant impact on risk among Asians, but significantly increased risk among non-Asians.ConclusionsThirty-day CW results in a measurable increase in epithelial permeability. Risk of inflammatory and lens-induced adverse events also increase over time during CW. Daily irrigation with sterile saline solution did not mitigate increases in epithelial permeability or decrease risk of adverse events.

Current cell-based assays for determining the functional properties of high-density lipoproteins (HDL) have limitations. We report here the development of a new, robust fluorometric cell-free biochemical assay that measures HDL lipid peroxidation (HDLox) based on the oxidation of the fluorochrome Amplex Red. HDLox correlated with previously validated cell-based (r = 0.47, p

BACKGROUND: Stress and shear force applied on blood components during processing and storage may induce cellular damage leading to release of cell-free DNA (cfDNA). In this study, we have compared ultraviolet (UV) spectrophotometry with UV-induced fluorescence for the quantification of cfDNA in red cell supernatant. MATERIALS AND METHODS: cfDNA was extracted from 200 μL sample of supernatants from 99 packed red blood cells using QIAamp DNA Blood Mini Kit (Qiagen, Germany). Quantification of cfDNA was done using two different methods: one based on spectrophotometry (NanoDrop 2000c, ThermoFisher Scientific, USA) and another based on fluorometry (Qubit 2.0, Life Technologies, ThermoFisher Scientific, USA). Interassay variability of both the methods was estimated using serial dilutions of standard with known DNA concentration. RESULTS: DNA quantification by both the methods was close to actual amount of known standard in dilutions with higher concentration of DNA (21.68 to 2.71 ng/μl). While at higher dilutions, quantification by NanoDrop was neither precise nor accurate. Median cfDNA concentration in the study units was found to be 1.60 ng/μl (25th–75th percentile range: 1.10–2.10) by UV spectrophotometry (NanoDrop) compared to 0.080 ng/μl (25th–75th percentile range: 0.050–0.130) by fluorometry (Qubit). CONCLUSION: Due to high interassay variability between the two methods and the better precision and accuracy of Qubit, it is recommended that fluorometry-based method be used for the quantification of cfDNA in blood components.

The methods of detection of mycotoxins in agricultural products have been analyzed. The advantages of using the fluorescence methods for the express diagnostics of the presence of mycotoxins in samples have been shown. The development of an optical biosensor, which allows the detection of mycotoxins in the field, has been presented. The principles of operation have been established, and the constructive solution of a sensor has been proposed. An electro-optical scheme for obtaining an information signal has been developed and tested. The particular attention has been paid to the choice of an element base of the proposed microsensor. The principles and procedure for its validation have been shown. The practical results of testing the developed technical solution have been presented. The achieved relative error in the linear approximation of the sensor conversion characteristics in the interval of concentrations 0-100 ppb at a temperature of 15-25 °C does not exceed 2%. The developed sensor can be used in agriculture for the express detection and evaluation of the mycotoxin contamination. [ABSTRACT FROM AUTHOR]

The lack of reproducibility of published studies is one of the major issues facing the scientific community, and the field of biofilm microbiology has been no exception. One effective strategy against this multifaceted problem is the use of minimum information guidelines. This strategy provides a guide for authors and reviewers on the necessary information that a manuscript should include for the experiments in a study to be clearly interpreted and independently reproduced. As a result of several discussions between international groups working in the area of biofilms, we present a guideline for the spectrophotometric and fluorometric assessment of biofilm formation in microplates. This guideline has been divided into 5 main sections, each presenting a comprehensive set of recommendations. The intention of the minimum information guideline is to improve the quality of scientific communication that will augment interlaboratory reproducibility in biofilm microplate assays.

PurposeTo ascertain whether a difference in the permeability of the corneal epithelium to fluorescein (Pdc) exists between Asians and non-Asians.MethodsFrom a multi-study database we extracted 632 records of baseline, open-eye Pdc measurements taken on both eyes of 176 subjects. Subjects were awake for a minimum of 4h before measurement, and were free of ocular disease and central corneal staining. Pdc was transformed by natural logarithm to better approximate normality for statistical tests.ResultsThe mean ln(Pdc) in the Asian group was significantly greater than in the non-Asian group [-2.34 ln(nm/s) vs. -2.58 ln(nm/s); p

This study focuses on understanding the structural and molecular changes in lipid membranes under the influence of six halogenated flavonoid derivatives differing in the number and position of substitution of chlorine and bromine atoms (D1-D6). Utilizing various analytical techniques, including fluorometric methods, dynamic light scattering (DLS), attenuated Fourier transform infrared spectroscopy (ATR- FTIR), and FT-Raman spectroscopy, the research aims to elucidate the mechanisms underlying the interaction of flavonoids with cell membranes. Additionally, the study includes in silico analyses to explore the physicochemical properties of these compounds and their potential pharmaceutical applications, along with toxicity studies to assess their effects on cancer, normal, and red blood cells. Our study showed the ability of halogenated derivatives to interact mostly with the outer part of the membrane, especially in the lipid heads region however, some of them were able to penetrate deeper into the membrane and affect the fluidity of hydrocarbon chains. The potential to reduce cancer cell viability, the lack of toxicity towards erythrocytes, and the favourable physicochemical and pharmacokinetic properties suggest these halogenated flavonoids potential candidates for exploring their potential for medical use., (© 2024. The Author(s).)

With the expansion of ICP-MS application into the field of bioanalysis, there is an urgent need for novel element tags today. Here, we report the design of a dual-element Ir-Eu tag, opening the door to simultaneous fluorescent imaging and ICP-MS quantification. The ratio of 153 Eu/ 193 Ir may serve as a precision control of the labeling process, allowing internal validation of the quantitative results obtained. As for SIRPα and its host cell analysis exemplified here, the Ir-Eu tag demonstrated superior figures of ICP-MS quantification with the LOD (3σ) down to 0.5 ( 153 Eu) and 1.1 ( 193 Ir) pM SIRPα and 220 ( 153 Eu) and 830 ( 193 Ir) RAW264.7 cells more than 130 times more sensitive compared with the LOD (3σ) of 65.2 pM SIRPα at 612 nm using fluorometry. Not limited to these demonstrations, we believe that the design ideas of the dual Ir-Eu tags should be applicable to various cases of bioanalysis when dual optical profiling and ICP-MS quantification are indispensable., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)