Human Plasma Samples Research Articles

Effect of Lactobacillus plantarum supplementation on trimethylamine-N-oxide levels in 30 patients with atherosclerotic cardiovascular disease: A double-blind randomized controlled trial

Introduction: Trimethylamine-N-oxide (TMAO) is a metabolite produced by intestinal microbiota. It is well recognized as an independent risk marker for cardiovascular and renal diseases and mortality. Aim: The aim of the present study was to investigate the effect of Lactobacillus plantarum GLP3 supplementation on TMAO levels in 30 patients with a history of atherosclerotic cardiovascular disease after 12 weeks of treatment. Materials and methods: Thirty consecutive male patients with a history of clinical atherosclerotic cardiovascular disease were randomized in the study. TMAO levels were evaluated in human plasma samples using high-performance liquid chromatography with triple quadrupole tandem mass spectrometry and results were presented as the median (interquartile range). Microbiome sequencing analysis, focusing on bacteria from the genus Lactobacillus, was performed in 21 patients. Results: Patients receiving probiotic treatment showed a significant decrease in the TMAO levels [from 284 (139) µg/L to 202.5 (96.7) µg/L; p=0.044], with no significant change apparent in the placebo group after the treatment [from 176 (120) µg/L to 178 (150) µg/L; p=0.258]. Lactobacillus spp. % in the probiotic group was significantly increased after the interventional procedures [0.6 (0.14) before and 0.79 (0.6) after the treatment; p=0.041]. Conclusion: Probiotic supplementation with Lactobacillus plantarum GLP3 reduced the TMAO levels in very high-risk patients for cardiovascular diseases.

Targeting an altered sphingolipid profile with consecutive lipotoxicity as therapeutic approach to calcific aortic valve disease

Abstract Background Aortic stenosis due to calcific aortic valve disease (CAVD) is the most common valvular heart disease. In symptomatic patients, without repair the prognosis is poor. Treatment to date is based on surgery or catheter-based interventions. Pharmacological therapy options to prevent the progression of valve calcification are not available. In vascular smooth muscle cells, cellular accumulation of sphingolipids is associated with a proinflammatory response in the sense of lipotoxicity. Chronic inflammatory processes are indeed also essential for aortic valve calcification. Purpose With the subsequent goal of developing innovative treatment approaches for CAVD, we therefore aim to elucidate the unclear role of sphingolipids in the development of CAVD. Methods Human aortic valve and plasma samples of CAVD patients and controls (n=26) were analysed by liquid chromatography–mass spectrometry for lipidomics and proteomics. Human aortic valve interstitial cells (hVICs) were stimulated with ceramides. Calcification was detected by alizarin red staining. mRNA expression of bone-related proteins was determined by qPCR. NF-kB pathway was assessed by proteome profiler. Myriocin and GW4869 were applied to inhibit ceramide biosynthesis. PDTC and MCC950 were used to inhibit NF-kB Pathway and NLRP3 activation. ApoE-/- mice received a gain-of-function PCSK9 adeno-associated virus vector and fed high cholesterol diet for 14 weeks. Myriocin was applied orally over the treatment period to inhibit ceramide de novo synthesis in vivo. Murine echocardiography was used to measure transvalvular velocity and left ventricular function. Calcification degree of murine aortic valve was determined by histological staining. Results A significantly altered sphingolipid profile was observed in aortic valve tissue of CAVD patients (Fig. 1). Ceramide species e.g. Cer (17:1;2O/16:0) were increased. Verifying the biological relevance of these findings in vitro, C2-Cer (d18:1/2:0) enhanced hVIC calcification (Fig. 2). Accordingly, ALP activity and mRNA expression of osteogenic genes RUNX2, ALPL and MSX2 were increased. Furthermore, C2-Cer (d18:1/2:0) enhanced NF-kB p65 phosphorylation and NLRP3 activation, while treatment with both PDTC and MCC950 protected against C2-Cer (d18:1/2:0) induced calcification. Supporting the role of sphingolipid biosynthesis in hVIC calcification, Myriocin and GW4869 both reduced ox-LDL-induced VIC calcification which was reversed by C2-Cer(d18:1/2:0). Finally, Myriocin reduced the degree of calcification and transvalvular jet velocity of aortic valve in the PCSK9-AAV ApoE-/- mice model. Conclusion CAVD is accompanied by an accumulation of ceramides causing lipotoxicity in human aortic valve tissue. Pharmacological modulation of sphingolipid metabolism is an effective approach to prevent the development and progression of aortic valve calcification in vivo and should be considered as a future therapeutic strategy in CAVD patients.Volcano Plot of CAVD tissue lipidomicsC2 Ceramide enhances hVIC calcification

A Simple HPLC-DAD Method for the Therapeutic Monitoring of Clozapine and Related Metabolites in Human Plasma and Urine Samples

Clozapine and its metabolites require close therapeutic monitoring (TDM) in patients due to poor correlation between the administrated doses and resulting plasma concentrations, the narrow therapeutic interval, high inter-individual variability, and the risk of serious side effects once toxic levels are exceeded. The aim of the study was to develop a simple (relatively cheap) LC-UV method for the quantification of clozapine and its metabolites in plasma and urine samples. For sample preparation, liquid-liquid extraction (LLE) in n-octanol was more efficient and less limiting in injection volumes compared to the in-situ formation of SUPRAS. When analyzing urine, an alkalinization step before extraction was required. The proposed method produced linear concentration responses with/without internal standard (IS) for the target analytes, with LLOQs within the targeted range of 50 ppb and %RSD within the acceptable 15% range. Furthermore, sample stability studies proved that pre-extracted samples were stable for the short term at room temperature and long-term when frozen.

Development of microflow ultra high performance liquid chromatography-mass spectrometry metabolomic assays for analysis of mammalian biofluids

Introduction and objectivesThe application of untargeted metabolomics assays using ultra high performance liquid chromatography-mass spectrometry (UHPLC-MS) to study metabolism in biological systems including humans is rapidly increasing. In some of these studies there is a requirement to collect and analyse low sample volumes of biofluids (e.g. tear fluid) or low cell and tissue mass samples (e.g. tissue needle biopsies). The application of microflow, capillary or nano liquid chromatography (≤ 1.0 mm column internal diameter (i.d.)) theoretically should accomplish a higher assay sensitivity compared to analytical liquid chromatography (2.1–5.0 mm column internal diameter). To date, there has been limited research into microflow UHPLC-MS assays that can be applied to study samples of low volume or mass.MethodsThis paper presents three complementary UHPLC-MS assays (aqueous C18 reversed-phase, lipidomics C18 reversed-phase and Hydrophilic Interaction Liquid Chromatography (HILIC)) applying 1.0 mm internal diameter columns for untargeted metabolomics. Human plasma and urine samples were applied for the method development, with porcine plasma, urine and tear fluid used for method assessment. Data were collected and compared for columns of the same length, stationary phase and stationary phase particle size but with two different column internal diameters (2.1 mm and 1.0 mm).Results and conclusionsAll three assays showed an increase in peak areas and peak widths when applying the 1.0 mm i.d. assays. HILIC assays provide an advantage at lower sample dilutions whereas for reversed phase (RP) assays there was no benefit added. This can be seen in the validation study where a much higher number of compounds were detected in the HILIC assay. RP assays were still appropriate for small volume samples with hundreds of compounds being detected. In summary, the 1.0 mm i.d. column assays are applicable for small volume samples where dilution is required during sample preparation.

Eco-Friendly Analytical Approach for Sensitive Spectrofluorimetric Determination of the Flavonoid Chrysin in Capsules and Human Plasma.

Chrysin is a plant flavonoid that has different therapeutic effects as anti-inflammatory, anti-cancer, anti-oxidant, and immune booster. Spectrofluorimetry has received a lot of interest lately because of itsecological greenness and analytical performance. This approach employed the native fluorescence of chrysin at 339nm following excitation at 231nm in distilled water. Modern advances in analytical chemistry have been used to lessen occupational and environmental concerns by employing distilled water as a dilution solvent throughmethod development and application. The approach was found to be excellent green supported byeco-scale score of 97and 0.94AGREErating, in addition to an overall whiteness score of 88.80. The design aimed to analyze chrysin in raw materials, Chrysin® capsules and human plasma. The method was linear over0.5-7.0ngmL⁻1 chrysin, with LOD of 0.06ngmL⁻1 and LOQ of 0.20ngmL⁻1. The offered methodwas effectively applied for determination of chrysin inthe commercial capsules Chrysin® and spiked human plasma samples with average recoveries of 99.76%and 99.98%, respectively for capsules andspiked human plasma. Up to date, no spectrofluorimetric method has been described for chrysin analysis, then, this presented an opportunity to develop a sensitive, quick, reliable, environmentally friendly, and valid fluorescence-based method.

Application of Recycled Battery-Graphite Electrode Decorated with Polyglutamic Acid/Au Nanoparticles for Detection of Nalbuphine Drug Abuse

Abstract We report a highly-uniform nanocomposite of polyglutamic acid (PGA) and gold nanoparticles (AuNPs) electrodeposited on a recycled battery graphite electrode (BGE) for the detection of Nalbuphine (NB), a semi-synthetic opioid. The sensor was optimized and characterized morphologically (via scanning electron microscopy, atomic force microscopy, and energy dispersive X-ray analysis) and electrochemically (via cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy). Under optimized conditions, the PGA/AuNPs/BGE revealed two linear ranges, 2.5×10-8 to1.0×10-6 M, and 2.0 × 10-6 to 1.0 × 10-4 M for Nalbuphine (NB), that is equivalent to 9.825×10-3 to 0.393 µg/mL and 0.786 to 39.30 µg/mL, with R2 = 0.995 and 0.994, respectively, and showed good catalytic activity for the determination of nalbuphine in the presence of tramadol and the oxidation potential of these opioid analgesic drugs were separated. The sensor was successfully applied for the detection of NB in its pharmaceutical formulations, spiked urine, and human plasma samples, without applying any sample pretreatment, at a recovery range of 99±0.03 to102±0.02% and thus, the developed can be considered as a promising approach for NB abuse testing in clinical and forensic agencies.

Simultaneous quantification of JWH-018, JWH-073, and their metabolites (4-OH JWH-018, 3-OH JWH-073) in human plasma and urine samples by LC-MS/MS

This study aims to develop and validate a method for the simultaneous quantification of JWH-073, JWH-018, and their two main metabolites 3-OH JWH-073 and 4-OH JWH-018 in human blood and urine samples by using liquid chromatography combined with a mass spectrometer (LC-MS/MS). The method was validated according to current FDA and EMA guidelines and was tested on 10 urine samples from suspected synthetic cannabinoid abusers and 10 plasma samples from drug-free volunteers. Experimental data showed that the limits of detection (LOD) for the analytes were very low at 0.01 ng/mL in plasma samples and 0.003 ng/mL in urine samples. The limits of quantification (LLOQ) were 0.03 ng/mL in plasma samples and 0.01 ng/mL in urine samples. The linear correlation coefficients (R 2) for these analytes ranged from 0.9914 to 0.9929 in plasma samples and 0.9820 to 0.9892 in urine samples. Among the 10 urine samples from suspected synthetic cannabinoid abusers, the parent JWH-073 and JWH-018 analytes were detected in six and three samples, with concentrations of 0.01 − 8.14 ng/mL and 7.16 − 17.50 ng/mL, respectively. At the same time, their two main metabolites, 3-OH JWH-073 and 4-OH JWH-018, were also found in the samples containing parent drugs, with concentrations of 32.13 − 84.24 ng/mL and 14.48–32.97 ng/mL, respectively. Three samples (T3, T5, and T6) contained only 4-OH JWH-018 metabolite (with concentrations of 25.69 − 51.87 ng/mL) and one sample (T10) contained only 3-OH JWH-073 metabolite (with concentration of 56.94 ng/mL) without the presence of their parent drugs. The plasma samples from drug-free volunteers were all negative for the analytes.

Fast Determination of Propofol in Human Plasma Using C18-Functionalized Magnetic Nanomaterials Followed by Supercritical Fluid Chromatography.

Novel C18-functionalized magnetic nanomaterials; i.e., C18@poly-styrene-divinylbenzene-glycidyl methacrylate-Fe3O4 (C18@PS-DVB-GMA-Fe3O4) have been synthesized by using N, N-dimethyloctadecylamine as modifying agent, which could be beneficial to remove the blood phospholipids. The C18@PS-DVB-GMA-Fe3O4 nanoparticles have been used and evaluated in the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) procedure for human plasma prior to the analysis of propofol by supercritical fluid chromatography (SFC). In the QuEChERS procedure, human plasma samples are directly mixed with extraction solvent and C18@PS-DVB-GMA-Fe3O4 nanoparticles, and the extraction and cleanup procedures have been accomplished simultaneously. The SFC separation was performed with a C18 column (Thermo Scientific™ Acclaim™ 120, 250 × 4. 6mm, 5μm) within 5min, using thymol as the internal standard. Supercritical carbon dioxide was used as the mobile phase with methanol as the cosolvent at the flow rate of 1.0mL/min. The column temperature was 38°C, and detection wavelength was 275nm. A good linearity was obtained among the propofol concentration range of 0.5-10mg/L (R2 = 0.9997) with the limit of detection of 0.17mg/L. Recoveries were in the range of 76.5-91.9%, with RSD less than 7.9%. These results suggested that method is convenient, rapid with high accuracy and little matrix effect, and suitable for rapid determination of propofol plasma concentration.

The Application of Untargeted Metabolomic Approaches for the Search of Common Bioavailable Metabolites in Human Plasma Samples from Lippia citriodora and Olea europaea Extracts.

Lippia citriodora and Olea europaea are known for their shared common bioactivities. Although both matrices are rich in similar families of bioactive compounds, their specific phytochemical compounds are mostly different. Since these compounds can be metabolized in the organism, this study hypothesized that common bioavailable metabolites may contribute to their similar bioactive effects. To test this, an acute double-blind intervention study in humans was conducted with blood samples collected at multiple time points. Using an untargeted metabolomic approach based on HPLC-ESI-QTOF-MS, 66 circulating metabolites were detected, including 9 common to both extracts, such as homovanillic acid sulfate and glucuronide derivates, hydroxytyrosol sulfate, etc. These common metabolites displayed significantly different Tmax values depending on the source, suggesting distinct metabolization pathways for each extract. The study highlights how shared bioavailable metabolites may underlie similar bioactivities observed between these two plant sources.

Glia dysfunction in schizophrenia: evidence of possible therapeutic effects of nervonic acid in a preclinical model.

Neuroinflammation may inhibit oligodendrocyte and astrocyte differentiation, which causes demyelination and synaptic degeneration. The myelin component nervonic acid (NA) may improve demyelinating and neurodegenerative diseases. This study firstly explored relationships between glial cell dysfunction and demyelination or synaptic degeneration in schizophrenia patients, and secondly determined nervonic acid therapeutic effects in a preclinical schizophrenia model of mice. Plasma samples were collected from 18 male healthy controls and 18 male schizophrenic patients (diagnosed by DSM-V) at aged 18-55. Mouse brain samples were collected from a maternal immune activation (MIA) model of schizophrenia via injecting 5mg/kg polyinosinic-polycytidylic acid. Male mouse offspring (age 2.5months, n = 12) were treated by clozapine (15mg/kg/day) or fed 0.5% NA for 6weeks. Cytokine and dopamine (DA) concentrations, and glial phenotypes and myelin markers were measured in both human plasma and mouse brain samples. In patient plasma, increased proinflammatory cytokines were associated with reactive microglia (Iba-1) up-regulation, while decreased anti-inflammatory cytokines were related to microglia (CD206) downregulation. Decreased astrocyte marker (p11) concentrations were accompanied by reduced concentrations of oligodendrocyte and synaptic markers. However, NA and DA contents were increased. Compared with control mice, SZ-like behaviors appeared in MIA male mice. Changes in microglia and astrocytes markers, and cytokine concentrations in the frontal cortex were consistent with those observed in patients' plasma. Hippocampal oligodendrocyte and synaptic marker expression were also decreased. DA content and DA/metabolite (DAPOC) were increased in MIA mouse brains. Most of these changes were normalized by both clozapine and NA. Even though some NA effects were more pronounced than clozapine, only clozapine restored cytokine function. The data suggest a possible therapeutic route for schizophrenia patients.

Utility of Microwave-Synthesized Silver Nano Particles as Spectrofluorimetric Sensors for the Determination of Nano Concentrations of Favipravir: Application to Dosage Forms and Spiked Human Plasma.

A simple and facile microwave-assisted method was developed for the synthesis of highly fluorescent silver-nanoparticles (Ag-NPs). The synthesis of silver-nanoparticles depends on a redox reaction between silver nitrate and ascorbic acid using chitosan as a stabilizing agent. The produced Ag-NPs were characterized using Zeta potential and transmission electron microscope micrograph where they are spherical in shape with smooth surface morphology and size of 26.81 ± 2.2nm. Favipiravir (FAV) was found to cause an obvious enhancement in the fluorescence of Ag-NPs; hence, they were used for its spectrofluorimetric estimation. The fluorescence intensity was measured at 430nm after excitation at 360nm. Under optimum conditions, a good linear relationship was accomplished between the FAV concentration and the fluorescence intensity in a range of (5.0-200.0) ng/mL with a limit of detection of 1.59 ng/mL. The method was successfully applied for the assay of the drug in its commercial tablets and spiked human plasma samples, and the results obtained were satisfactory.

A Cost‐Effective Electrochemical Sensor Using a Graphite Pencil Electrode for Sensitive Quantification of Phenolic Antioxidants in Human Plasma and Food Samples

AbstractThe pressing challenge in electroanalysis revolves around the creation of a user‐friendly analytical sensor that is accessible to individuals lacking specialized expertise. Such sensor should offer minimal cost, while simultaneously ensuring high sensitivity and reproducibility. Herein, graphite pencil electrode (GPE) has emerged as a promising electrochemical sensor for the determination of polyphenolic compounds, including gentisic acid (GEN), gallic acid (GA), caffeic acid (CAF), and sinapic acid (SA). To comprehensively explore the electrochemical oxidation processes of these antioxidants, a range of electrochemical techniques, namely cyclic voltammetry (CV), differential pulse voltammetry (DPV), and chronoamperometry (CA), were deployed. The results of these investigations unveiled a diffusional oxidation wave associated with phenolic hydroxyl groups, involving a two‐electron/two‐proton loss step, occurring within the potential range of 0.4–0.6 V vs. Ag/AgCl. Crucially, the GPE exhibited the capacity to establish linear calibration curves for GEN, GA, CAF, and SA, encompassing concentrations from nanomolar to sub‐micromolar which highlighted by the corresponding limits of detection (LoD), recorded at 0.086, 0.046, 0.112, and 0.115 μM, respectively. The heightened sensitivity and reproducibility demonstrated by the GPE underscore its potential as an efficient tool for the determination of polyphenolic compounds. Beyond its analytical capabilities, the GPE displayed remarkable applicability in facilitating on‐site, cost‐effective determinations of polyphenolic compounds in both plasma and food samples. This versatility positions the GPE as a valuable asset in addressing the challenges associated with electrochemical analysis, making it an attractive option for a wide range of applications where simplicity, affordability, and reliability are paramount.

Effective Determination of Diosmin Using Nitrogen Doped Carbon Dots as Probe Based on Internal Filtering Effect.

The establishment of a convenient and efficient testing method is crucial and needed for the monitoring of diosmin. In this study, nitrogen doped carbon dots (N-CDs) with the particle size distribution of 2.5-5.7nm and the average diameter of 4.1nm were successfully synthesized using a simple strategy. N-CDs exhibited excellent and stable fluorescence performance with the quantum yield of 22.33%. Correspondingly, a fluorescence analytical method was developed for diosmin determination using N-CDs as probe. UV-vis absorbance spectroscopy and the evaluation of internal filtering parameters verified that the mechanism causing the quenching of N-CDs was an internal filtering effect (IFE). The concentration of diosmin can be directly evaluated based on the quenched fluorescence intensities. After optimizing experimental conditions, it was found that the fluorescence quenching efficiency ((F0-F)/F0) of N-CDs exhibited a good linear relationship with the concentration of diosmin (CDiosmin) in the range of 3.0-50µg/mL. The limit of detection (LOD) was 0.86µg/mL based on 3σ/slope (n = 13). This method was successfully applied to accurately determine the content of diosmin in diosmin tablets and human plasma samples with good reproducibility. It stands out for its simplicity, speed, and acceptable determination performance.

Long-term stability of sufentanil quantified by UPLC-MS-MS in human plasma frozen for 11 years at -20°C.

The long-term stability of drug concentrations in human plasma samples when stored under normal laboratory conditions over several years is important for research purposes and clinical re-evaluation, but also for forensic toxicology. Fifty human plasma samples from a former clinical trial were re-analyzed after storage at -20°C for 11 years. Plasma samples were extracted using solid-phase extraction. Isotope labelled sufentanil-D5 was used as internal standard. Sufentanil plasma concentrations were determined by ultra-performance liquid chromatography (UPLC) with gradient elution, followed by tandem mass spectrometry with electrospray ionization. The linear dynamic range (LDR) was 25 - 2500 pg/mL, the limit of detection was 10 pg/mL, and the lower limit of quantification was 25 pg/mL. Intra- and inter-assay error did not exceed 6%. The deviation of the measured sufentanil plasma concentrations between the reanalysis and the first analysis was -63 ± 14% (mean ± SD). Therefore, sufentanil concentrations in human plasma were not stable in samples frozen at -20°C over 11 years.

Electrochemical Analysis of Amyloid Plaques and ApoE4 with Chitosan-Coated Gold Nanostars for Alzheimer's Detection.

Monitoring the progression of Alzheimer's disease (AD) is crucial for mitigating dementia symptoms, alleviating pain, and improving mobility. Traditionally, AD biomarkers like amyloid plaques are predominantly identified in cerebrospinal fluid (CSF) due to their concentrated presence. However, detecting these markers in blood is hindered by the blood-brain barrier (BBB), resulting in lower concentrations. To address this challenge and identify pertinent AD biomarkers-specifically amyloid plaques and apolipoprotein E4 (ApoE4)-in blood plasma, we propose an innovative approach. This involves enhancing a screen-printed carbon electrode (SPCE) with an immobilization matrix comprising gold nanostars (AuNSs) coated with chitosan. Morphological and electrical analyses confirmed superior dispersion and conductivity with 0.5% chitosan, supported by UV-Vis spectroscopy, cyclic voltammetry, and Nyquist plots. Subsequent clinical assays measured electrical responses to quantify amyloid-β 42 (Aβ42) (15.63-1000 pg/mL) and APoE4 levels (0.41 to 40 ng/mL) in human blood plasma samples. Differential pulse voltammetry (DPV) responses exhibited peak currents proportional to biomarker concentrations, demonstrating high linear correlations (0.985 for Aβ42 and 0.919 for APoE4) with minimal error bars. Cross-reactivity tests with mixed solutions of amyloid-β 40 (Aβ40), Aβ42, and ApoE4 indicated minimal interference between biomarkers (<3% variation), further confirming the high specificity of the developed sensor. Validation studies demonstrated a strong concurrence with the gold-standard enzyme-linked immunosorbent assay (ELISA), while interference tests indicated a minimal variation in peak currents. This improved device presents promising potential as a point-of-care system, offering a less invasive, cost-effective, and simplified approach to detecting and tracking the progression of AD. The substantial surface binding area further supports the efficacy of our method, offering a promising avenue for advancing AD diagnostics.

The mouse monoclonal antibody 4E3 is specific for the G1m17 allotype of human IgG1

Allotype is an amino acid variation within the immunoglobulin isotypes. Four allotypes have been described for human IgG1 and two of them (G1m3 and G1m17) are located at position 214 in the CH1 region of the gamma chain. Various diseases have been associated with allotype expression, making the allotype research an interesting field in immunology. However, allotype-specific reagents are rare. In the present study the specificity of a widely used and commercially available IgG1-specific monoclonal antibody named 4E3, described as binding to the hinge region of IgG1, was evaluated. Using the ImmunoCAP™ assay technology, surprisingly no IgG1 was measured in 13 of 23 human serum and plasma samples when 4E3 was used in an antibody-enzyme conjugate as detection reagent. Further evaluation of 4E3 using eight IgG1 myeloma paraproteins revealed that 4E3 did not bind to three of them. No association was seen between the binding pattern and myeloma light chain glycosylation or the kappa or light chain use. By comparing the myeloma paraprotein binding patterns of 4E3 and TM14 (a monoclonal antibody with known G1m3 specificity), it was indicated that 4E3 only bound to myeloma paraproteins expressing the G1m17 allotype. This was confirmed using recombinant human antibodies expressing either the G1m3 or G1m17 allotype. The G1m17 bias of 4E3 seen using ImmunoCAP was also observed in microtiter plate-based enzyme-linked immunosorbent assays. The antibody 4E3 has a G1m17 bias limiting its use in assays to measure IgG1 antibodies. However, it may be a valuable allotype-specific reagent.

Development of pseudotyped VSV-SARS-CoV-2 spike variants for the assessment of neutralizing antibodies.

Aim: Serological studies with pseudotyped viruses offer a safer alternative to live SARS-CoV-2 in evaluating neutralizing antibodies, enabling research in standard labs.Methods: The SARS-CoV-2 Spike pseudotyped vesicular stomatitis virus (VSV) pseudoviruses were generated using Spike of Wuhan strain and two variants (B.1.1.7, B.1.351) and utilized to evaluate the serum neutralizing activity of human plasma samples of vaccinated (n=13) and healthy people (n=2) compared with a plaque assay with authentic virus.Results: Neutralizing titer of convalescent plasma resulted with a good correlation (R2=0.7).Conclusion: We evaluated a safe and reliable pseudotyped virus system that effectively mimics authentic virus and correlates well with traditional assays. The developed system allows easier testing of variants and has the potential to improve vaccine development.

Fusion of Raman and FTIR Spectroscopy Data Uncovers Physiological Changes Associated with Lung Cancer.

Due to the high mortality rate, more effective non-invasive diagnostic methods are still needed for lung cancer, the most common cause of cancer-related death worldwide. In this study, the integration of Raman and Fourier-transform infrared spectroscopy with advanced data-fusion techniques is investigated to improve the detection of lung cancer from human blood plasma samples. A high statistical significance was found for important protein-related oscillations, which are crucial for differentiating between lung cancer patients and healthy controls. The use of low-level data fusion and feature selection significantly improved model accuracy and emphasizes the importance of structural protein changes in cancer detection. Although other biomolecules such as carbohydrates and nucleic acids also contributed, proteins proved to be the decisive markers found using this technique. This research highlights the power of these combined spectroscopic methods to develop a non-invasive diagnostic tool for discriminating lung cancer from healthy state, with the potential to extend such studies to a variety of other diseases.

A Lean Six Sigma approach aided improvement for simultaneous electrochemical determination of antiviral drug favipiravir and levodropropizine as an option in the treatment of COVID-19 using glassy carbon electrode modified NiFe2O4 nanocomposite in different matrices

Favipiravir (FPV) is a widely used drug that works well against a variety of infectious RNA viruses. It is commonly used as an anti-influenza medication. To treat patients infected with RNA viruses, combination therapy consisting of FPV and levodropropizine (LDP) is required. As a result, an effective electrochemical sensor for identifying FPV in human plasma samples has been developed. The hydrothermal method is used to synthesize NiFe2O4NPs, a binary transition metal oxide comprising iron and nickel. The effective synthesis of these materials is verified by energy-dispersive X-ray analysis (EDX) and X-ray diffraction (XRD). The developed nanocomposite underwent physicochemical and electrochemical characterization, and the experimental setup was refined. Two broad linear dynamic ranges were obtained for both medications using Differential Pulse Voltammetry (DPV): 0.5 µM–200 µM for FPV and 1.0 µM–110 µM for LDP. For FPV, the computed detection and quantitation limits were 0.148 µM and 0.45 µM, while for LDP, they were 0.297 µM and 0.9 µM. The recommended technique was successfully used to analyze both medications at the same time in human plasma samples, and acceptable percentage recoveries were attained. In terms of greenness, EcoScale and GAPI greenness tools were used to evaluate the analytical methodology. Additional studies on repeatability, stability testing, interference analysis, and greenness evaluation supported the validity of the recommended method for the simultaneous measurement of FPV and LDP in laboratories for quality assurance. Additionally, Lean Six Sigma (LSS) quality technique is regarded as a collection of management disciplines for continuous improvement and method control. It is thought to offer the most practical way to address the negative effects of variation by taking steps toward analytical process improvement. As a result, LSS can be considered an invaluable tool in any laboratory, serving as a standard method for process optimization and problem solutions as well as a managerial discipline. Furthermore, it guarantees a strong method specification at a high degree of statistical confidence and targeted performance.

Comparative Impact of Organic Grass-Fed and Conventional Cattle-Feeding Systems on Beef and Human Postprandial Metabolomics-A Randomized Clinical Trial.

Cattle-feeding systems may have health implications for consumers of beef products. Organic grass-fed (GRA) and conventional (CON) cattle-feeding systems may result in beef products with differing metabolite profiles and therefore could impact the postprandial metabolomic response of consumers. This study aims to measure whole beef metabolomics and postprandial metabolomic response of consumers between GRA and CON beef to elucidate potential health implications. This study followed a randomized double-blind crossover design with healthy male and female subjects (n = 10). Plasma samples were taken at fasting (0) and postprandially for four hours after consumption of a steak from each condition. Untargeted metabolomic analysis of whole beef and human plasma samples used LC/MS. Multivariate and pathway enrichment analysis in MetaboAnalyst was used to investigate metabolite and biochemical pathways that distinguished CON and GRA. Cattle-feeding systems impacted both postprandial and whole beef steak metabolomic profiles. Metabolites that contributed to this variation included carnitine species (Proionylcarnitine), fatty acids, amino acids (L-valine), and Calamendiol. These metabolites have been associated with oxidative stress, inflammation, and cardiovascular health. Functional pathway enrichment analysis revealed numerous amino acid degradation pathways, especially branched-chain amino acids, and fatty acid degradation that changed throughout the postprandial time course. These findings suggest that CON and GRA cattle-feeding systems differentially impact whole beef metabolomics, as well as consumer postprandial metabolic responses and the associated health implications.