Concentration Range Research Articles

Easy-to-Engineer Flexible Nanoelectrode Sensor from an Inexpensive Overhead Projector Sheet for Sweat Neuropeptide-Y Detection.

In this paper, we report an inexpensive and easy-to-engineer flexible nanobiosensor electrode platform by exploring a nonconductive overhead projector (OHP) sheet for sweat Neuropeptide-Y (NPY) detection, a potential biomarker for stress, cardiovascular regulation, appetite, etc. We converted a nonconductive OHP sheet into a conductive nanobiosensor electrode platform with a hybrid polymerization method, which consists of interfacial polymerization of pyrrole and a template-free electropolymerization technique to decorate the electrode platform with poly(EDOT-COOH-co-EDOT-EG3) nanotubes. The selection of poly(EDOT-COOH) features an easy conjugation of NPY antibody (NPY-Ab) through EDC/Sulfo-NHS coupling chemistry, while poly(EDOT-EG3) is best known to reduce nonspecific binding of biomolecules. The antibody conjugation on the polymer surface was characterized by a quartz crystal microbalance, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and chronoamperometry techniques. The OHP nanosensor platform exhibited the successful detection of NPY analyte through a chronoamperometry method in phosphate-buffered saline with a wide range of concentrations from 1 pg/mL to 1 μg/mL with a limit of detection of 0.68 pg/mL having good linearity (R2 = 0.9841). The sensor platform exhibited excellent stability, reproducibility, repeatability, and a shelf-life of 13 days. Furthermore, the sensor showed superior selectivity to a 100 pg/mL NPY analyte among other interfering compounds such as tumor necrosis factor α, cortisol, and Interleukin-6. The clinical practicality of the sensor was confirmed through the detection of 100 pg/mL NPY spiked artificial perspiration, highlighting the possibility of integrating the sensor platform to wearable healthcare applications.

Potential biological applications of environment friendly synthesized iron oxide nanoparticles using Sageretia thea root extract.

The green synthesis of Iron oxide nanoparticles (IONPs) has shown numerous advantages over conventional physical and chemical synthesis methods as these methods non-ecofriendly and uses toxic chemicals and complicated equipments. In present study, Iron oxide nanoparticles (IONPs) were created using simple, sustainable, eco-friendly and green chemistry protocol. The roots of novel medicinal plant Sageretia thea was used as a bio-template for the preparation of IONPs. Further, the synthesis of IONPs was confirmed using different analytical tools like UV-Vis, FT-IR, XRD, EDX, and SEM. The average sizes of (NPs) were found to be 16.04nm. Further, asynthesized IONPs were evaluated for several biological potentials including antibacterial, antifungal Anti-radical potentials (DPPH) and cytotoxicity assays. Antibacterial potencies were investigated using bacterial strains (in the concentration range of 1000-31.25µg/mL) revealing significant antibacterial potentials. ABA and SAU was reported to be least susceptible while KPN was observed to be most susceptible strain in bactericidal studies. Further, different fungal strains were used to investigate the antifungal potentials of IONPs (in the concentration range of 1000-31.25µg/mL) and revealed strong antifungal potencies against different pathogenic strains. Furthermore, MRA, FA and ANI were most susceptible and ABA was least susceptible in fungicidal examination. Significant cytotoxicity potential was examined using brine shrimps cytotoxicity assay, thus revealing the cytotoxic potential of asynthesized IONPs. The IC50 for S. thea based IONPs was recorded as 33.85µg/mL. Strong anti-radical potentials (DPPH) assay was performed to evaluate the ROS scavenging potential of S.T@IONPs. The highest scavenging potential was noted as 78.06%, TRP as 81.92% and TAC as 84% on maximum concentration of 200µg/mL. In summary, our experimental results concluded, that asynthesized IONPs have strong antibacterial, antifungal, DPPH scavenging and cytotoxic potentials and can be used in different biological applications. In nutshell, our as-prepared nanoparticles have shown potential bioactivities and we recommend, different other in vitro and in vivo biological and bioactivities to further analyze the biological potentials.

Copper and zinc status in cord blood and breast milk and child's neurodevelopment at 18months: Results of the Italian PHIME cohort.

Trace elements, including zinc (Zn) and copper (Cu), although toxic at higher concentrations are known to play important roles in the maintenance of human health and neurodevelopment. Few epidemiological studies have investigated the association between prenatal or early postnatal Cu and Zn levels and child neurodevelopment. The aim of this research is to assess the association between child neurodevelopment at 18 months of age and cord blood and breast milk concentrations of Cu and Zn in Italian mother-child pairs enrolled in the Italian Northern Adriatic Cohort II (NAC-II), a part of the "Public health impact of long-term, low-level, mixed element exposure in susceptible population strata" project PHIME. The study population consisted of 632 children, and their mothers, born within the NAC-II, who were tested with the Bayley Scales of Infant and Toddler Development third edition (BSID-III) at age 18 months. Cu and Zn concentrations were measured in cord blood and breast milk samples. Only children born at term (≥37 gestational week), who completed the BSID-III test and had at least 1 measure of Cu and Zn concentrations were included in the analysis. Information about socio-demographics and lifestyles were collected through questionnaires at different phases of follow-up. Cu and Zn concentrations were log2 transformed because of their skewed distribution. Multiple linear regression models were performed to study the association between each BSID-III composite score (cognitive, motor and language) and each metal concentration. Separate models were applied for each biological sample. The β coefficient (β) and its 95% confidence interval (95% CI) were estimated. Stratified analyses by child's sex were also conducted. The mean±standard deviation (SD) of cognitive, motor and language composite scores were respectively: 106±8, 101±5 and 97±8. The mean±SD of Cu and Zn concentrations (ng/g) were respectively 699.2±129.0 and 2538±589 in cord blood and 607±498 and 3226±1428 in breast milk. No association between metal concentration and cognitive composite score was found. A higher motor composite score was associated with higher Cu concentrations in cord blood (β=4.31 95% CI 2.03; 6.59). No associations were found between language composite score and metal concentrations. The effect of Cu cord blood concentration on motor composite score was confirmed when stratified by sex: males (β=5.49 95% CI 2.15; 8.36) and females (β=3.11; 95% CI 0.00; 6.22). A direct association, in females only, was found between language composite score and Cu concentration in cord blood (β=5.60 95% CI 0.63; 10.57) and in breast milk (β=3.04 95% CI 1.06; 5.03), respectively. The results from this cohort study showed a strong direct association between prenatal Cu levels and child motor neurodevelopment at 18 months. However, for generalizability, future research on the effects of Zn and Cu on neurodevelopment should include a larger range of early-life concentration of trace elements.

Toxicological assessment of the effects of CuCl2 and CuO nanoparticles on early developmental stages of the South American toad, Rhinella arenarum by standardized bioassays.

The release in aquatic environments of emergent contaminants such as copper oxide nanoparticles (CuO-NPs) has generated concerns on their short- and long-term toxicity and the potential risk for more vulnerable animal groups, such as amphibians. In this sense, the aim of this work was to evaluate the toxicity of CuO-NPs in comparison with its respective salt (CuCl2) in embryos and larvae of a native amphibian, Rhinella arenarum, by acute (96h) and chronic (504h) standardized bioassays. Lethality and sublethal effects such as developmental, morphological, and ethological alterations were assessed in a wide range of concentrations (0.001-100mg/L). Neurotoxic effects by acetyl (AChE) and butyrylcholinesterase (BChE) activity levels and changes in the lipid content were also assessed at sublethal concentrations. Results showed that CuCl2 caused higher lethality than CuO-NPs in both developmental periods. Embryos were more sensitive than larvae with LC50-96h = 0.080mg CuCl2/L and 1.26mg CuO-NPs/L and 0.21mg CuCl2/L and 20.17mg CuO-NPs/L, respectively. At acute exposure, embryos exhibited several developmental abnormalities such as developmental delay, edema, axial flexure, and microcephaly. Larvae presented spasmodic contractions and weak movements. Regarding neurotoxicity, a significant increase in AChE activity at low concentrations as well as an inhibition of BChE activity at all tested concentrations was evidenced for both substances at acute exposure. Moreover, an increment in phospholipid and triglyceride levels was observed at the highest concentration of CuO-NPs (10mg/L) at chronic exposure. The chromatographic separation of lipids showed no apparent differences in acylglycerols and free fatty acid bands, between the treatments and the control. The differences in toxicity between CuO-NPs and CuCl2 could be due to structural and physicochemical characteristics that influence their bioavailability and toxicity. Considering the exponential growth in the production and use of these substances, it is expected that the levels of contamination will rise considerably in the future, so that wildlife, particularly aquatic organisms, will be more increasingly exposed, representing a potential risk for their populations.

A novel miniaturized potentiometric electrode based on carbon nanotubes and molecularly imprinted polymer for the determination of lidocaine.

A novel miniaturized, solid-contact potentiometric screen-printed electrode was developed for highly sensitive and selective determination of lidocaine anesthetic. The electrode integrated single-walled carbon nanotubes as a solid-contact material and a molecularly imprinted polymer as a recognition sensory material. The performance characteristics of the electrode were evaluated and optimized to display a Nernstian slope of 58.92 ± 0.98mV/decade over a linear concentration range of 4.53 × 10-7 to 6.18 × 10-3mol/l within < 6s. Thedetection limit was 7.75 × 10-8mol/l (18.16ng/ml) of lidocaine. The use of the molecularly imprinted polymer significantly enhanced the selectivity of the electrode, and carbon nanotubes increased the sensitivity, accuracy, and potential stability. The electrode was successfully used for determining lidocaine in pharmaceutical preparations and human urine. The results favorably compared with data obtained by liquid chromatography-tandem mass spectrometry.

Machine Learning Insights into Ascorbic Acid-Enhanced Germination of Black Cumin (Nigella sativa L.) under Cadmium Stress

AbstractThe objective of the present work is to study the impact of seed priming with varying concentrations of ascorbic acid (vitamin C) on the germination process of black cumin (Nigella sativa) under cadmium (Cd) stress. As expected, Cd had a great effect on germination rates and seedling growth. However, the application of ascorbic acid during seed priming effectively alleviated Cd stress and significantly increased seed vigor. Primed seeds exhibited markedly elevated final germination percentage, germination index, mean germination time, seedling length, seedling vigor index, and reduced abnormal seedling percentage. Additionally, vitamin priming reduced membrane lipid peroxidation, in treated seeds. Moreover, seed priming elicited a considerable increase in peroxidase and catalase activity, thus mitigating stress effects and augmenting seed vitality. Our experimental data allowed us to establish 100–150 mg/L as the optimal concentration range for ascorbic acid in seed priming of black cumin. These insights were further corroborated through modeling techniques based on supervised machine learning. Notably, XGBoost emerged as a proficient tool for predicting final germination percentage, mean germination time, seedling vigor index, abnormal seedling percentage, and peroxidase activity, while SVR demonstrated aptitude in forecasting catalase activity and germination index. The Gaussian method exhibited superior performance in predicting malondialdehyde content. These comprehensive findings substantiate the premise that vitamin priming with ascorbic acid serves as a promising strategy to ameliorate germination outcomes under Cd-induced stress conditions.

Sequential fermentation of Ginkgo biloba seeds by Bacillus subtilis natto and Lactobacillus plantarum enhanced nutrition, flavor and lipid-lowering activity.

Ginkgo biloba seeds (GBS) are rich in flavonoids, proteins and reducing sugar, and have been consumed as food and medicinal nuts for thousands of years. However, the presence of ginkgotoxins and their poor palatability limit people's consumption of them. This study used solid-state fermentation with Bacillus subtilis natto and Lactobacillus plantarum to enhance the safety and benefits of GBS. Optimized fermentation conditions increased the content of beneficial components like total flavonoids, soluble protein and reducing sugar while eliminating unpleasant odors (isoamyl aldehyde and hexanal) and reducing the toxin 4'-O-methylpyridoxine by 91.17%. Fermentation of GBS powder can significantly enhance its anti-inflammatory and antioxidant activities in vitro (P < 0.001). Furthermore, it exhibits a dose-dependent effect within a certain concentration range. Mixed fermentation (FBnLp) was evaluated for its effects on obesity and metabolic syndrome in mice fed a high-fat diet. FBnLp significantly reduced body and liver weight gain, prevented dyslipidemia and decreased inflammatory and oxidative stress compared to unfermented GBS. Histological analysis showed that FBnLp improved liver health by reducing fat accumulation and preventing non-alcoholic fatty liver disease. Meanwhile, it was found that feeding FBnLp increased the expression of CPT-1α, which regulates energy expenditure and fat breakdown, and downregulated the expression of SREBP-1c, FAS and ACC, which regulate fat synthesis. This research provides new insights and technological support for the application and development of FBnLp as a functional product, addressing key issues in its use and industry growth. © 2024 Society of Chemical Industry.

Development and Validation of Rapid and Cost-Effective RP-HPLC/UV Method for Determination of Cetirizine/Levocetirizine in Blood Plasma of Smokers and Non Smokers: a Two Cohort Comparative Study.

In this research study, a simple, rapid, sensitive, and cost-effective Reverse Phase High Performance Liquid Chromatography method was developed and validated for determination of Cetirizine and Levocetirizine in human plasma. The drugs were separated on an ACE Generix100-5 C18 RP (250 × 4.6mm, 5μm) column, using Acetonitrile and Distilled water pH7 (59:41v/v) as mobile phase with a flow rate of 1.5mL/min, retention time of 3.39 ± 0.02min, detection wavelength of 230nm, and column oven temperature at 26°C. The drug was extracted from human plasma using Methanol, the percent recovery was 97%. The LOD and LOQ for Cetirizine and Levocetirizine were 0.5ng and 1ng. The method was linear over the concentration range of 0.5-1000ng /mL for Cetirizine and Levocetirizine. The method was applied for analysis of drugs in plasma following oral administration of Cetirizine (10mg) and Levocetirizine (5mg) in smokers and nonsmokers of Pashtun ethnicity belonging from different districts of Khyber Pakhtunkhwa, Pakistan. The difference between Cetirizine plasma levels in smokers and non-smokers was significant with p -value 0.0279.

Microplastics Meet Metoprolol in Natural Water: Sorption Behavior and Mechanism

As an ideal carrier for the spread of pollutants in the aquatic environment, microplastics (MPs) can adsorb pharmaceutical β-blockers, which can affect their migration and lead to some unpredictable adverse consequences. In this paper, the sorption behaviors and mechanism of MPs (polyvinyl chloride (PVC) and polypropylene (PP)) for typical β-blocker metoprolol (MTL) were investigated. The effects of pH, salinity and humic acids (HAs) on the sorption were studied, which proved that the sorption behavior was different under different environmental conditions. Both low pH and high salinity inhibited the sorption of MTL by the MPs. Specifically, the sorption capacity of MTL increased, with pH increase from 3 to 10. When pH = 10, the sorption capacities of MTL on PVC (1.75 mg/g) and PP (3.34 mg/g) reached the maximum. After pH &gt; 10, the amount of MTL adsorbed on PVC was slightly decreased, while that on PP was essentially the same. The addition of salt ions inhibited the sorption in the concentration range of 5–250 mg/g for both NaCl and CaCl2, with the inhibitory effect of Ca2+ being stronger than that of Na+. Moreover, the presence of HAs promoted the sorption of MPs for MTL. In the absence of HAs, the sorption capacities of PP and PVC for MTL were 0.34 mg/g and 0.79 mg/g, respectively. When HA concentration was 100 mg/L, the highest sorption capacities of PP and PVC reached 0.79 mg/g and 1.37 mg/g, respectively. This indicated that the promoting effect of HAs on PP was stronger than that on PVC. In general, based on the study of the sorption behavior of MTL and the characterization of the MPs, the sorption mechanism was speculated to consist mainly of electrostatic interactions, cation exchange, hydrophobic interaction and halogen bonding. The sorption kinetics of MTL on the two MPs were well-fitted by the pseudo-second-order model with R2 &gt; 0.99. The sorption isotherms both fitted the Freundlich model, which substantiated that the sorption of MTL on the MPs (PVC and PP) was multilayered and heterogeneous. Collectively, these findings provided a theoretical basis for revealing the complex interactions between MPs and MTL in natural water and a new insight into the fate and migration of MPs and β-blockers in the environment.

Copper homeostasis and neurodegenerative diseases

Copper, one of the most prolific transition metals in the body, required for normal brain physiological activity and allows various functions to work normally through its range of concentrations. Copper homeostasis is meticulously maintained through a complex network of copper-dependent proteins, including copper transporters (CTR1 and CTR2), the two copper ion transporters the Cu -transporting ATPase 1 (ATP7A) and Cu-transporting beta (ATP7B), and the three copper chaperones ATOX1, CCS, and COX17. Disruptions in copper homeostasis can lead to either the deficiency or accumulation of copper in brain tissue. Emerging evidence suggests that abnormal copper metabolism or copper binding to various proteins, including ceruloplasmin and metallothionein, is involved in the pathogenesis of neurodegenerative disorders. However, the exact mechanisms underlying these processes are not known. Copper is a potent oxidant that increases reactive oxygen species production and promotes oxidative stress. Elevated reactive oxygen species levels may further compromise mitochondrial integrity and cause mitochondrial dysfunction. Reactive oxygen species serve as key signaling molecules in copper-induced neuroinflammation, with elevated levels activating several critical inflammatory pathways. Additionally, copper can bind aberrantly to several neuronal proteins, including alpha-synuclein, tau, superoxide dismutase 1, and huntingtin, thereby inducing neurotoxicity and ultimately cell death. This study focuses on the latest literature evaluating the role of copper in neurodegenerative diseases, with a particular focus on copper-containing metalloenzymes and copper-binding proteins in the regulation of copper homeostasis and their involvement in neurodegenerative disease pathogenesis. By synthesizing the current findings on the functions of copper in oxidative stress, neuroinflammation, mitochondrial dysfunction, and protein misfolding, we aim to elucidate the mechanisms by which copper contributes to a wide range of hereditary and neuronal disorders, such as Wilson's disease, Menkes' disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and multiple sclerosis. Potential clinically significant therapeutic targets, including superoxide dismutase 1, D-penicillamine (DPA), and 5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline (PBT2), along with their associated therapeutic agents, are further discussed. Ultimately, we collate evidence that copper homeostasis may function in the underlying etiology of several neurodegenerative diseases and offer novel insights into the potential prevention and treatment of these diseases based on copper homeostasis.

First direct measurements of the nitrogen isotopic composition of vehicle tailpipe-emitted NH3 and implications for the source apportionment of NH3 in urban atmosphere.

The introduction of three-way catalytic converter (TWC) to meet stringent vehicular NOx emission standards worldwide has led to an unintended consequence of vehicle-derived ammonia (NH3) emission, which might degrade air quality and affect human health, especially in urban areas. The nitrogen stable isotope composition of NH3 (δ15N-NH3) may be a useful tool to trace NH3 sources, but the isotopic signature of vehicle-emitted NH3 is lacking. Here we report the δ15N-NH3 measured from tailpipe exhausts collected directly from 19 different vehicles equipped with TWC using "grab" sample technique optimized to avoid isotopic fractionation. We found a large of range of NH3 concentration (from 1 to 37 ppm, x̄ ± 1σ = 10 ± 8 ppm) and δ15N value (from -38.1 ‰ to 49.0 ‰, x̄ ± 1σ = 1.2 ± 20.9 ‰) for our samples (n = 57). Our results indicate a correlation between δ15N-NH3 and NH3 concentrations (i.e. δ15N-NH3 (‰) = 16.9(± 2.2)In(NH3 concentration; ppm) - 31.7(± 4.7) (R2 = 0.53, P < 0.01) that is associated with the catalyst temperature, which is generally agree with the results of theoretical calculation. After consideration of the vehicle emission evolution, dominant driving condition and tunnel δ15N-NH3 variation, a representative δ15N-NH3 value (meanminmax) of vehicle source is identified as 0.8000-2.20005.2000, which is distinct from (larger than) other NH3 volatilization-related emission sources. Using our newly measured δ15N values and Bayesian mixing model, we approximate the overall contribution of vehicles to ambient NH3 before, during, and after an international event in Beijing was 14.1 %, 7.9 %, and 21.5 %, respectively, which agree well with the course of traffic restriction. Collectively, our findings demonstrate the importance of using vehicle-emitted δ15N-NH3 to quantify vehicular NH3 contribution in urban atmospheres.

Glucocorticoids influence on rat hematological parameters and catalase activity

In this study, the impact of glucocorticoid, betamethasone dipropionate on enzyme activity in vitro and its effects on hematological parameters in vivo was investigated. The immobilized catalase, crucial for cell oxidative stress response via hydrogen peroxide reduction, exhibited a robust electrocatalytic response, maintaining its biological activity. The in vitro inhibition kinetics of catalase, as determined by electrocatalytic methods and expressed using Lineweaver-Burke diagrams, revealed an uncompetitive type of inhibition with altered Imax and Km in the presence of a range of betamethasone dipropionate concentrations. The in vivo experiments conducted on Rattus norvegicus demonstrated significant alterations in hematological parameters following betamethasone dipropionate administration. These changes included a decrease in erythrocyte count, an increase in hemoglobin, a reduction in mean corpuscular volume (MCV), and an elevation in mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC). Notably, the leukocyte counts substantially increased. The observed hematological shifts suggest an impact of betamethasone dipropionate on the hematopoietic system, reinforcing the need for cautious corticosteroid administration. The findings underline the necessity for judicious corticosteroid treatment, acknowledging both enzymatic and systemic repercussions.

Deciphering supramolecular arrangements, micellization patterns, and antimicrobial potential of bacterial rhamnolipids under extreme treatments of temperature and electrolyte

The micellization properties of rhamnolipids (RLs) in extreme electrolyte concentrations and temperatures have gained considerable attention due to their broad industrial applications. In this study, the aggregation behavior, specifically the micellization pattern (critical micelle concentration (CMC)) of RLs produced from a newly isolated thermophilic strain of Pseudomonas aeruginosa from a harsh environment of an oil field, was investigated by a spectrophotometric method at various temperatures (293–393 K) and electrolyte concentrations (NaCl: 2–20%). The result indicated that the CMC values (0.267–0.140 mM⋅dm−3) were both electrolyte- and temperature-dependent exhibiting a U-shaped trend as temperature and NaCl concentration increased. Variations in NaCl concentration and temperature also affected the standard Gibbs free energy (ΔGomic), enthalpy (ΔHomic), and entropy (ΔSomic) of micellization. The molecule also showed stability at a broad range of temperatures, pH, and NaCl concentrations. Thin-layer chromatography (TLC) and Fourier-transform infrared (FTIR) analysis confirmed the similarity in composition between the crude extract and the commercial RL with Rf values of 0.72 for mono-rhamnolipids and 0.28 for di-rhamnolipids. FTIR analysis confirmed the chemical nature particularly key aliphatic functional groups present in the fatty acid tail of RLs and the -COC- bond in the structure of the rhamnose moiety. Additionally, LC-ESI-QTOF analysis confirmed corresponding ionic fragments of mono- and di-rhamnolipids congeners. Furthermore, the antimicrobial potential was determined against different human pathogens in the absence and presence of NaCl by measuring zones of inhibition. The result revealed enhanced inhibitory effects against Gram-positive pathogens (S. aureus, S. epidermidis, and L. monocytogene), with zones of inhibition of 26, 30, and 20 mm in the presence of NaCl. These findings underline the role of NaCl in the micellization of RL molecules and highlight their importance in environmental applications, pharmaceuticals, and various life science sectors.

The novel mechanism on the inhibition of superoxide anion generating from xanthine oxidase in the presence of myricetin.

To gain an in-depth understanding of the process of myricetin (MY) inhibiting the generation of •O2- from Xanthine Oxidase (XOD) at a novel perspective, the inhibiting pathway is necessary to be re-elaborated through inhibitory type, thermodynamic analysis and molecular simulation. The results demonstrated that MY is indeed a potent inhibiting agent for •O2- producing from XOD, with the maximum value of 33.78 % in the inhibition of •O2-. Furthermore, the deprotonated form (De-MY) of MY was examined to be an effective agent for interacting with XOD, with the measurement of •O2- in productions from MY, XOD@MY and XAN-XOD@MY, as comparing to the XAN-XOD@ALL. On the inhibition kinetics, De-MY was witnessed to be on a mixed-type inhibition, with more competitive inhibition. In the thermodynamic analysis, the Stern-Volmer plots for De-MY obtained at 298 K, 303 K and 308 K, were polynomial-fitted to form curves approaching the X axis via the fluorescence quenching of XOD, which was against the reported with good linearity. It indicated that the interaction between De-MY and XOD occurred at the range of MY concentrations from 0 to 0.025 (×10-5 mol L-1), is dominated by a static quenching procedure, ascribing to the appearance of accessible interaction with corresponding f values less than 1. As well, via combining the synchronous fluorescence data, Molecular dynamics simulations showed that MY binding to the Trp 283, ARG-60 and Tyr 58 with H-binding interactions is an available access to block the transfer of free electrons from FADH2 to O2. Therefore, in this work, successfully re-elaborating the inhibition mechanism of •O2- generating from XOD by MY is benefited for MY in the future applications.

Glucose measurements in sheep using a long-term continuous glucose monitor designed for humans

This study evaluated the feasibility of utilizing a continuous glucose monitors (CGM) designed for use in humans to measure glucose levels in sheep. Four Suffolk x Dorset sheep were fitted with jugular catheters and FreeStyle Libre 2 (Abbott®) glucose monitors. Glucose concentration from the CGM were compared with those from a glucometer and traditional assays during a hyperglycemic clamp, aiming to explore a broader range of physiological glucose concentrations in a controlled manner. Measurements were taken every five minutes during the infusion and every ten minutes post-infusion until baseline levels were restored. Relationships were analyzed using a linear mixed-effects model with glucometer readings as the response variables, GCM reading as fixed effects, and animal as random effect with significant level of p &amp;lt; 0.05. The results demonstrated a significant linear correlation between the CGM and glucometer readings both during the infusion (p = 0.0003) and afterward (p = 0.006). A glucose calorimetric test was used to confirm glucose concentrations on samples and used as gold standard. Although the glucometer and CGM did not differ from the assay method, they did differ from one another (p = 0.045). Upon more in-depth analysis, the random intercepts for animal were highly significant and one CGM showed values numerically much higher than other CGM and other glucose analysis methods. No difference among methods was observed (p = 0.715) when the outlier animal was omitted. While promising, CGM demands confirmation of initial readings and standardization against established methods before wider adoption in research or clinical applications.

Abstract A012: Analytical validation of a cfDNA-based fragmentomic profiling assay for the detection and quantification of 14 HPV types in cervical cancer

Abstract Purpose: Human papillomavirus (HPV) is the primary cause of cervical cancer, one of the most common cancers affecting women globally. Despite advances in prevention and screening, there remains a critical need for reliable, non-invasive assays to detect and monitor HPV-driven cervical cancers. Liquid biopsy offers a promising solution to meet this need. Here, we report the analytical validation of a plasma-based fragmentomic profiling assay designed to detect and characterize circulating tumor tissue modified viral (TTMV)-HPV DNA from 14 HPV types associated with cervical cancer. Methods: A high-complexity, laboratory-developed test was developed that employs 68 primers and 34 probes to target 14 HPV types via droplet digital PCR (ddPCR). A quantitative algorithm was applied to integrate fragment counts to generate a TTMV-HPV DNA Score, reflective of the quantity of circulating tumor-derived HPV DNA. Analytical validation was performed by determining the limit of blank (LOB), limit of quantitation (LOQ), and limit of detection (LOD) for each HPV type. To measure the limits of assay performance, engineered samples were used for reference interval studies. Synthesized target-specific DNA regions of HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV66, and HPV68 were diluted to varying concentrations to perform assay validation. Results: The assay demonstrated excellent specificity, with LOBs ranging from 0 to 0.07 copies/µL across the 14 HPV types. LODs ranged from 0.20 to 1.02 copies/µL, indicating a high level of sensitivity. The assay also showed high accuracy and precision, with coefficients of variation (CV) below 20% for all HPV types across both intra- and inter-assay precision studies. Regression analysis confirmed strong linearity (R2 &amp;gt; 0.99) across a wide range of analyte concentrations. Conclusion: These results demonstrate that TTMV-HPV DNA assay accurately and reproducibly detects circulating HPV DNA for 14 HPV types commonly associated with cervical cancer, offering a valuable tool for both diagnosis and ongoing surveillance in clinical settings. Future studies will present the clinical validation in order to establish the prognostic utility of the assay in predicting the likelihood of HPV-driven cervical cancer in patients. Citation Format: Sunil Kumar, Joshua Hutcheson, David Conway, Michael Horan, Syandan Chakraborty, Chloe Wiseman, Alicia Gunning, Evgeny Skyrypkin, Cassin Williams, Catherine Del Vecchio Fitz, Piyush B Gupta. Analytical validation of a cfDNA-based fragmentomic profiling assay for the detection and quantification of 14 HPV types in cervical cancer [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A012.

Studies on the ssDNA-Based Biosensor Regeneration and Miniaturization for Electrochemical Detection of miRNAs

Abstract One of the most significant disadvantages of biosensing systems is the limited possibility of their regeneration, which only allows for their single use for detection of most targets. The reduction of biosensor fabrication cost could thus be achieved by elaboration of protocol providing the highest recovery of sensing layer response. A further drop of production expenses could yield the biosensor miniaturization as it leads to consumption of chemicals required for receptor layer formation as well as execution of measurements. To address the above-mentioned challenges, we aimed to find the most adequate method of regeneration of single-stranded DNA-based layers specific to miRNA 141 molecule which elevated concentration might refer to progression of cancer. The studies indicated that 5 min. incubation of ssDNA-modified electrode in 4 M urea provided the highest response towards miRNA 141 among all tested regeneration procedures. Furthermore, the possibility of ssDNA immobilization on was shown. This enabled miRNA 141 detection within 0.1 nM – 1 µM concentration range with high selectivity. Moreover, ssDNA layers elaborated on miniaturized transducers were distinguished with sufficient stability after 24 h storage in 20 mM PBS and could be also regenerated using 4 M urea.

Effect of Aminophenol and Amino Acids Derivatives on the Level of Nitrosyl Radical and Its Active Forms &lt;i&gt;In Vitro&lt;/i&gt;

The effect of aminophenol, amino acids and their derivatives on the level of •NO and its intermediates (•NO2, N2O3) in an aqueous aerobic media (pH = 7.4) was studied using nitroprusside as a •NO donor. It was found that the highest NOx scavenging activity is exhibited by 3-aminophenol (IC50 = 0.11 mM), 2-aminophenol (IC50 = 0.195 mM) and 4,6-di-tert-butyl-2-aminophenol (IC50 = 0.12 mM), standards: trolox (IC50 = 0.19 mM) and ascorbate (IC50 = 4.88 mM). Methylation of the OH group reduced the effectiveness of aminophenol. In the studied concentration range (0–70 mM), Tyr-Ala (IC50 = 5.0 mM) and β-Ala-His (IC50 = 35.0 mM) were more active than Phe-Ala (IC50 50 mM) and Gly-Gly (IC50 50 mM). Complexes Cu(Gly)2 and Cu(Gly-Gly)2 at low concentrations (0.05–0.5 mM) are 1.4–1.8 times more effective than Gly and Gly-Gly.

Novel analytical approach for baclofen quantification in rodent plasma.

A simple, accurate, and robust reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the quantification of Baclofen in rat plasma. The method demonstrated high degree of linearity (r2 = 0.9936) across a concentration range of 10-50 μg/mL. Precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), and robustness were evaluated according to ICH guidelines. The LOD and LOQ were found to be 0.076197 and 0.23090 μg/mL, respectively. This method provides an efficient approach for Baclofen quantification in plasma, making it suitable for pharmacokinetic and bioavailability studies. The novelty of this study lies in its optimization for routine use in laboratories, ensuring reproducibility with minimal variations across different conditions and analysts.

The mammalian Ire1 inhibitor, 4µ8C, exhibits broad anti-Aspergillus activity in vitro and in a treatment model of fungal keratitis

ObjectiveThe fungal unfolded protein response consists of a two-component relay in which the ER-bound sensor, IreA, splices and activates the mRNA of the transcription factor, HacA. Previously, we demonstrated that hacA is essential for Aspergillus fumigatus virulence in a murine model of fungal keratitis (FK), suggesting the pathway could serve as a therapeutic target. Here we investigate the antifungal properties of known inhibitors of the mammalian Ire1 protein both in vitro and in a treatment model of FK.MethodsThe antifungal activity of Ire1 inhibitors was tested against conidia of several A. fumigatus isolates by a broth microdilution assay and against fungal biofilm by XTT reduction. The influence of 4μ8C on hacA mRNA splicing in A. fumigatus was assessed through gel electrophoresis and qRT-PCR of UPR regulatory genes. The toxicity and antifungal profile of 4μ8C in the cornea was assessed by applying drops to uninfected or A. fumigatus-infected corneas 3 times daily starting 4 hours post-inoculation. Corneas were evaluated daily through slit-lamp imaging and optical coherence tomography, or at endpoint through histology or fungal burden quantification via colony forming units.ResultsAmong six Ire1 inhibitors screened, the endonuclease inhibitor 4μ8C displayed the strongest antifungal profile with an apparent fungicidal action. The compound both blocked conidial germination and hyphal metabolism of A. fumigatus Af293 in the same concentration range that blocked hacA splicing and UPR gene induction (60-120 µM). Topical treatment of sham-inoculated corneas with 0.5 and 2.5 mM 4μ8C did not impact corneal clarity, but did transiently inhibit epithelialization of corneal ulcers. Relative to vehicle-treated Af293-infected corneas, treatment with 0.5 and 2.5 mM drug resulted in a 50% and &amp;gt;90% reduction in fungal load, respectively, the latter of which corresponded to an absence of clinical signs of infection or corneal pathology.ConclusionThe in vitro data suggest that 4μ8C displays antifungal activity against A. fumigatus through the specific inhibition of IreA. Topical application of the compound to the murine cornea can furthermore block the establishment of infection, suggesting this class of drugs can be developed as novel antifungals that improve visual outcomes in FK patients.