Human Samples Research Articles

Dual Sensing of Dopamine and Acetaminophen Mediated by Biomass Derived Phosphorous Rich Carbon

AbstractMultimolecular detection using a single electrode matrix as well as utilization of inexpensive non‐metal doped carbon from biomass is an attractive approach to developing low‐cost sensors. Herein, we report the synthesis of phosphorous‐doped carbon (PDC) from almond seed skin by a simple self‐polymerization method and utilized as a sensing matrix for Dopamine (DA) and Acetaminophen (APAP) whose redox potential is nearer. The phytic acid in the almond seed skin provides phosphorus to the carbon during high‐temperature treatment. The elemental analysis results confirm the existence of phosphorous in PDC. The sensing results illustrate that the PDC modified glassy carbon electrode (PDC/GCE) exhibits excellent performance against DA and APAP with a wide linear range (0.5 μM–10 μM). The detection limit (LOD) and sensitivity have been calculated and the value is found to be 0.130 μM, 0.047 μA μM−1 for DA and 0.335 μM, 0.021 μA μM−1 for APAP, respectively. The sensitivity of PDC/GCE in real sample conditions was measured using DA and APAP spiked human serum and plasma samples, the results indicate excellent recoveries ranging from 96.00 % to 103.10 %. These encouraging findings reveal that the PDC is an appropriate material for determining the DA and APAP.

TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

Immune memory has been expanded to group 2 innate lymphoid cells (ILC2s), but the cellular and molecular bases remain incompletely understood. Based on house dust mite (HDM)-induced mice asthma models and human samples, we applied flow cytometry, parabiosis, in vivo imaging and adoptive transplantation to confirm the persistence, migration and function of CD45+lineage–CD90.2+NK1.1–NKp46–ST2–KLRG1+IL-17RB+ memory-like ILC2s (ml-ILC2s). Regulated by CCR9/CCL25 and S1P signaling, ml-ILC2s reside in the lamina propria of small intestines (siLP) in asthma remission, and subsequently move to airway upon re-encountering antigens or alarmins. Furthermore, ml-ILC2s possess properties of longevity, potential of rapid proliferation and producing IL-13, and display transcriptional characteristics with up-regulation of Tox and Tcf-7. ml-ILC2s transplantation restore the asthmatic changes abrogated by Tox and Tcf7 knockdown. Our data identify siLP ml-ILC2s as a memory-like subset, which promotes asthma relapse. Targeting TCF-1 and TOX might be promising for preventing asthma recurrence.

Highly accurate and sensitive absolute quantification of bacterial strains in human fecal samples

BackgroundNext-generation sequencing (NGS) approaches have revolutionized gut microbiome research and can provide strain-level resolution, but these techniques have limitations in that they are only semi-quantitative, suffer from high detection limits, and generate data that is compositional. The present study aimed to systematically compare quantitative PCR (qPCR) and droplet digital PCR (ddPCR) for the absolute quantification of Limosilactobacillus reuteri strains in human fecal samples and to develop an optimized protocol for the absolute quantification of bacterial strains in fecal samples.ResultsUsing strain-specific PCR primers for L. reuteri 17938, ddPCR showed slightly better reproducibility, but qPCR was almost as reproducible and showed comparable sensitivity (limit of detection [LOD] around 104 cells/g feces) and linearity (R2 > 0.98) when kit-based DNA isolation methods were used. qPCR further had a wider dynamic range and is cheaper and faster. Based on these findings, we conclude that qPCR has advantages over ddPCR for the absolute quantification of bacterial strains in fecal samples. We provide an optimized and easy-to-follow step-by-step protocol for the design of strain-specific qPCR assays, starting from primer design from genome sequences to the calibration of the PCR system. Validation of this protocol to design PCR assays for two L. reuteri strains, PB-W1 and DSM 20016 T, resulted in a highly accurate qPCR with a detection limit in spiked fecal samples of around 103 cells/g feces. Applying our strain-specific qPCR assays to fecal samples collected from human subjects who received live L. reuteri PB-W1 or DSM 20016 T during a human trial demonstrated a highly accurate quantification and sensitive detection of these two strains, with a much lower LOD and a broader dynamic range compared to NGS approaches (16S rRNA gene sequencing and whole metagenome sequencing).ConclusionsBased on our analyses, we consider qPCR with kit-based DNA extraction approaches the best approach to accurately quantify gut bacteria at the strain level in fecal samples. The provided step-by-step protocol will allow scientists to design highly sensitive strain-specific PCR systems for the accurate quantification of bacterial strains of not only L. reuteri but also other bacterial taxa in a broad range of applications and sample types.-oGjUmj5e8MXZDxyDjzcm-Video

MiRNA-193a-mediated WT1 suppression triggers podocyte injury through activation of the EZH2/β-catenin/NLRP3 pathway in children with diabetic nephropathy

Diabetic nephropathy (DN), an eminent etiology of renal disease in patients with diabetes, involves intricate molecular mechanisms. Recent investigations have elucidated microRNA-193a (miR-193a) as a pivotal modulator in DN, although its precise function in podocyte impairment remains obscure. The present study investigated the role of miR-193a in podocyte injury via the WT1/EZH2/β-catenin/NLRP3 pathway. This study employed a comprehensive experimental approach involving both in vitro and in vivo analyses. We utilized human podocyte cell lines and renal biopsy samples from pediatric patients with DN. The miR-193a expression levels in podocytes and glomeruli were quantified via qRT‒PCR. Western blotting and immunofluorescence were used to assess the expression of WT1, EZH2, β-catenin, and NLRP3 inflammasome components. Additionally, the study used luciferase reporter assays to confirm the interaction between miR-193a and WT1. The impact of miR-193a manipulation was observed by overexpressing WT1 and inhibiting miR-193a in podocytes, followed by analysis of downstream pathway activation and inflammatory markers. We found upregulated miR-193a in podocytes and glomeruli, which directly targeted and suppressed WT1, a crucial podocyte transcription factor. WT1 suppression, in turn, activated the EZH2/β-catenin/NLRP3 pathway, leading to inflammasome assembly and proinflammatory cytokine production. Overexpression of WT1 or inhibition of miR-193a attenuated these effects, protecting podocytes from injury. This study identified a novel mechanism by which miR-193a-mediated WT1 suppression triggers podocyte injury in DN via the EZH2/β-catenin/NLRP3 pathway. Targeting this pathway or inhibiting miR-193a may be potential therapeutic strategies for DN.

The optimization and validation of a gas chromatography-mass spectrometry method to analyze the concentration of acetate, propionate and butyrate in human plasma or serum

Fermentation-derived short-chain fatty acids (SCFA)44SCFA: short-chain fatty acids. are potential mediators of the health benefits associated with dietary fiber intake. SCFA affect physiological processes locally in the gut and on distant organs via the systemic circulation. Since SCFA are used as energy source for colonocytes and substrate for the liver metabolism, their concentrations in the systemic circulation are low. Therefore, quantification of systemic SCFA requires sensitive analytical techniques. This article covers the optimization and validation of a gas chromatography-mass spectrometry method to measure systemic SCFA concentrations following derivatization with 2,4-difluoroaniline (DFA)55DFA: 2,4-difluoroaniline. and extraction in ethyl acetate. Sample preparation was optimized by varying the amount of DFA, coupling agent 1,3-dicyclohexylcarbodiimide, ethyl acetate and sodium bicarbonate, which is used to quench derivatization. In addition, evaporation of the samples using a vacuum concentrator resulted in less contamination, notably of acetate, compared to drying with N2 gas. The method showed excellent linearity with coefficient of variation (R2) > 0.99 and a good precision (relative standard deviation < 20 %) and accuracy. Finally, systemic concentrations of SCFA in human plasma samples could successfully be determined.

Functional Analysis of RE1 Silencing Transcription Factor as a Putative Tumor Suppressor in Human Endometrial Cancer.

Uterine cancer is the most common gynecologic malignancy in the United States, with endometrioid endometrial adenocarcinoma (EC) being the most common histologic sub-type. Considering the molecular classifications of EC, efforts have been made to identify additional biomarkers that can assist in diagnosis, prognosis, and individualized therapy. We sought to explore the relationship of Repressor Element 1 (RE1) silencing transcription factor (REST), which downregulates neuronal genes in non-neuronal tissue, along with matrix metalloproteinase-24 (MMP24) and EC. We analyzed the expression of REST and MMP24 in 31 cases of endometrial cancer and 16 controls. We then explored the baseline expression of REST and MMP24 in two EC cell lines (Ishikawa and HEC-1-A) compared to a benign cell line (t-HESC) and subsequently evaluated proliferation, migration, and invasion in the setting of loss of REST gene expression. REST and MMP24 expression were significantly lower in human EC samples compared to control samples. REST was highly expressed in EC cell lines, but decreasing REST gene expression increased proliferation (FC: 1.13X, p < 0.0001), migration (1.72X, p < 0.0001), and invasion (FC: 7.77X, p < 0.05) in Ishikawa cells, which are hallmarks of cancer progression and metastasis. These findings elicit a potential role for REST as a putative tumor suppressor in EC.

Molecularly Imprinted Polypyrrole-Modified Screen-Printed Electrode for Dopamine Determination.

This paper introduces a quantitative method for dopamine determination. The method is based on a molecularly imprinted polypyrrole (e-MIP)-modified screen-printed electrode, with differential pulse voltammetry (DPV) as the chosen measurement technique. The dopamine molecules are efficiently entrapped in the polymeric film, creating recognition cavities. A comparison with bare and non-imprinted polypyrrole-modified electrodes clearly demonstrates the superior sensitivity, selectivity, and reproducibility of the e-MIP-based one; indeed, a sensitivity of 0.078 µA µM-1, a detection limit (LOD) of 0.8 µM, a linear range between 0.8 and 45 µM and a dynamic range of up to 350 µM are achieved. The method was successfully tested on fortified synthetic and human urine samples to underline its applicability as a screening method for biomedical tests.

An efficient chemiluminescent probe based on Ni-doped CsPbBr3 perovskite nanocrystals embedded in mesoporous SiO2 for sensitive assay of L–cysteine

This study presents an efficient chemiluminescence (CL) probe based on perovskite nanocrystals (NCs) for detection of L–cysteine (L–Cys). It consists of nickel–doped CsPbBr3 NCs embedded in the mesoporous SiO2 matrix as CL reagent and cerium (IV) as an oxidant in aqueous environment. The probe was designed for the highly selective determination of L–Cys based on its remarkable enhancing effect on the CL intensity. The colloidal nanocomposite of nickel–doped CsPbBr3 NCs@SiO2 with photoluminescence quantum yield of 58% was fabricated by ligand–assisted re–precipitation method and characterized by using UV–Vis absorption, FT–IR, X–ray diffraction, and transmission electron microscopy. The sensor was utilized to determine L–Cys in the linear concentration range of 20–300 nM with a detection limit of 12.8 nM. Direct chemical oxidation of Ni–doped CsPbBr3 NCs@SiO2 by Ce(IV) was the single cause of the formation of the excited-state NCs and subsequent production of CL. The developed probe provides outstanding selectivity towards L–Cys over structurally related compounds. Accurate determination of L–Cys in human serum samples was achieved without interference, and the results were confirmed by HPLC method.

The rs3918188 and rs1799983 loci of eNOS gene are associated with susceptibility in patients with systemic lupus erythematosus in Northeast China

To investigate the association between single nucleotide polymorphism (SNP) at the rs3918188, rs1799983 and rs1007311 loci of the endothelial nitric oxide synthase (eNOS) gene and genetic susceptibility to systemic lupus erythematosus (SLE) in northeastern China. The base distribution of eNOS gene rs3918188, rs1799983 and rs1007311 in 1712 human peripheral blood samples from Northeast China was detected by SNaPshot sequencing technology. The correlation between genotype, allele and gene model of these loci of the eNOS gene and the genetic susceptibility to SLE was investigated by logistic regression analysis. The results of the differences in the frequency distribution of their gene models were visualised using R 4.3.2 software. Finally, HaploView 4.2 software was used to analyse the relationship between the haplotypes of the three loci mentioned above and the genetic susceptibility to SLE. A multifactor dimensionality reduction (MDR) analysis was used to determine the best SNP-SNP interaction model. The CC genotype and C allele at the rs3918188 locus may be a risk factor for SLE (CC vs AA: OR = 1.827, P < 0.05; C vs A: OR = 1.558, P < 0.001), and this locus increased the risk of SLE in the dominant model and the recessive model (AC + CC vs AA: OR = 1.542, P < 0.05; CC vs AA + AC: OR = 1.707, P < 0.001), while the risk of SLE was reduced in the overdominant model (AC vs AA + CC: OR = 0.628, P < 0.001). The GT genotype and T allele at locus rs1799983 may be a protective factor for SLE (GT vs GG: OR = 0.328, P < 0.001; T vs G: OR = 0.438, P < 0.001) and this locus reduced the risk of SLE in the overdominant model (GT vs GG + TT: OR = 0.385, P < 0.001). There is a strong linkage disequilibrium between the rs1007311 and rs1799983 loci of the eNOS gene. Among them, the formed haplotype AG increased the risk of SLE compared to GG. AT and GT decreased the risk of SLE compared to GG. In this study, the eNOS gene rs3918188 and rs1799983 loci were found to be associated with susceptibility to SLE. This helps to deeply explore the mechanism of eNOS gene and genetic susceptibility to SLE. It provides a certain research basis for the subsequent exploration of the molecular mechanism of these loci and SLE, as well as the early diagnosis, treatment and prognosis of SLE.

Deep Learning Powers Protein Identification from Precursor MS Information.

Proteome analysis currently heavily relies on tandem mass spectrometry (MS/MS), which does not fully utilize MS1 features, as many precursors remain unselected for MS/MS fragmentation, especially in the cases of low abundance samples and wide abundance dynamic range samples. Therefore, leveraging MS1 features as a complement to MS/MS has become an attractive option to improve the coverage of feature identification. Herein, we propose MonoMS1, an approach combining deep learning-based retention time, ion mobility, detectability prediction, and logistic regression-based scoring for MS1 feature identification. The approach achieved a significant increase in MS1 feature identification based on an E. coli data set. Application of MonoMS1 to data sets with wide dynamic range, such as human serum proteome samples, and with low sample abundance, such as single-cell proteome samples, enabled substantial complementation of MS/MS-based peptide and protein identification. This method opens a new avenue for proteomic analysis and can boost proteomic research on complex samples.

RAD52 resolves transcription-replication conflicts to mitigate R-loop induced genome instability

Collisions of the transcription and replication machineries on the same DNA strand can pose a significant threat to genomic stability. These collisions occur in part due to the formation of RNA-DNA hybrids termed R-loops, in which a newly transcribed RNA molecule hybridizes with the DNA template strand. This study investigated the role of RAD52, a known DNA repair factor, in preventing collisions by directing R-loop formation and resolution. We show that RAD52 deficiency increases R-loop accumulation, exacerbating collisions and resulting in elevated DNA damage. Furthermore, RAD52’s ability to interact with the transcription machinery, coupled with its capacity to facilitate R-loop dissolution, highlights its role in preventing collisions. Lastly, we provide evidence of an increased mutational burden from double-strand breaks at conserved R-loop sites in human tumor samples, which is increased in tumors with low RAD52 expression. In summary, this study underscores the importance of RAD52 in orchestrating the balance between replication and transcription processes to prevent collisions and maintain genome stability.

Formaldehyde quantification using gas chromatography–mass spectrometry reveals high background environmental formaldehyde levels

Formaldehyde (HCHO) is a human toxin that is both a pollutant and endogenous metabolite. HCHO concentrations in human biological samples are reported in the micromolar range; however, accurate quantification is compromised by a paucity of sensitive analysis methods. To address this issue, we previously reported a novel SPME–GC–MS-based HCHO detection method using cysteamine as an HCHO scavenger. This method showed cysteamine to be a more efficient scavenger than the widely used O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine, and enabled detection of aqueous HCHO in the nanomolar range and quantification in the micromolar range. However, quantification in this range required immersive extraction of the HCHO-derived thiazolidine, while a high background signal was also observed. Following on from these studies, we now report an optimised head-space extraction SPME–GC–MS method using cysteamine, which provides similarly sensitive HCHO quantification to the immersive method but avoids extensive wash steps and is therefore more amenable to screening applications. However, high background HCHO levels were still observed A Complementary GC–MS analyses using a 2-aza-Cope-based HCHO scavenger also revealed high background HCHO levels; therefore, the combined results suggest that HCHO exists in high (i.e. micromolar) concentration in aqueous samples that precludes accurate quantification below the micromolar range. This observation has important implications for ongoing HCHO quantification studies in water, including in biological samples.

Long-term persistence of diverse clones shapes the transmission landscape of invasive Listeria monocytogenes

BackgroundThe foodborne bacterium Listeria monocytogenes (Lm) causes a range of diseases, from mild gastroenteritis to invasive infections that have high fatality rate in vulnerable individuals. Understanding the population genomic structure of invasive Lm is critical to informing public health interventions and infection control policies that will be most effective especially in local and regional communities.MethodsWe sequenced the whole draft genomes of 936 Lm isolates from human clinical samples obtained in a two-decade active surveillance program across 58 counties in New York State, USA. Samples came mostly from blood and cerebrospinal fluid. We characterized the phylogenetic relationships, population structure, antimicrobial resistance genes, virulence genes, and mobile genetic elements.ResultsThe population is genetically heterogenous, consisting of lineages I–IV, 89 clonal complexes, 200 sequence types, and six known serogroups. In addition to intrinsic antimicrobial resistance genes (fosX, lin, norB, and sul), other resistance genes tetM, tetS, ermG, msrD, and mefA were sparsely distributed in the population. Within each lineage, we identified clusters of isolates with ≤ 20 single nucleotide polymorphisms in the core genome alignment. These clusters may represent isolates that share a most recent common ancestor, e.g., they are derived from the same contamination source or demonstrate evidence of transmission or outbreak. We identified 38 epidemiologically linked clusters of isolates, confirming eight previously reported disease outbreaks and the discovery of cryptic outbreaks and undetected chains of transmission, even in the rarely reported Lm lineage III (ST3171). The presence of animal-associated lineages III and IV may suggest a possible spillover of animal-restricted strains to humans. Many transmissible clones persisted over several years and traversed distant sites across the state.ConclusionsOur findings revealed the bacterial determinants of invasive listeriosis, driven mainly by the diversity of locally circulating lineages, intrinsic and mobile antimicrobial resistance and virulence genes, and persistence across geographical and temporal scales. Our findings will inform public health efforts to reduce the burden of invasive listeriosis, including the design of food safety measures, source traceback, and outbreak detection.

A nanohybrid-based smartphone-compatible high performance electrochemical glucose sensor

A cost-effective and low-potential smartphone-compatible electrochemical sensor was constructed using a nanohybrid for the sensing of glucose in human and animal serum. The nanohybrid composed of gold nanoparticle (GNP) decorated cobalt hexacyanoferrate (CHCF) modified ZIF-67 (cobalt metal organic framework, CMOF) was synthesized and characterized by FTIR, XRD, and SEM with EDX analysis. The electrochemical characteristics of the nanohybrid were investigated by depositing the nanohybrid over a conventional glassy carbon electrode (GCE) and performing cyclic voltammetry, which revealed a stable redox peak with a formal potential of +0.23 V, corresponding to Co2+/3+ redox couple in GNP–CHCF–CMOF. Thus, developed sensor was utilized for the electrochemical glucose detection, which showed exceptional electrocatalytic activity over a linear detection range from 8.33 to 3793 μM with a low detection limit of 0.96 μM at a low potential of +0.35 V. Furthermore, the GNP–CHCF–CMOF/GCE sensor was employed for the detection of glucose spiked in human and rabbit serum samples, which showed excellent recoveries. A portable measurement device was fabricated which showed the real-time monitoring of glucose in a smartphone. This novel approach paves the way for the design and fabrication of cost-effective, low-potential sensors, which would reduce overall costs and enhance the performance of sensing devices.

An Electrochemical Nucleic Acid Biosensor for Triple-Negative Breast Cancer Biomarker Detection.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, affecting younger women and women of minorities. The nomenclature "triple negative" is derived from the absence of the three most common breast cancer biomarkers: progesterone receptor (PR), estrogen receptor (ER), and human epidermal growth factor receptor 2 (HER2). It derives its name from testing negative for these three most common breast cancer biomarkers. Currently, TNBC is diagnosed at advanced stages, necessitating the need for a diagnostic tool or method to identify this malignancy at an early stage prior to metastasis. In this study, a novel electrochemical biosensor was developed, optimized, and evaluated for the detection of microRNA-10b (miRNA-10b), marking the first use of this biomarker for the early diagnosis of TNBC. The biosensor demonstrated the ability to detect concentrations as low as 10 pM. Furthermore, the biosensor was specific toward the target biomarker, distinguishing non-target miRNAs of similar size. The efficacy of the biosensor for TNBC early diagnosis was further validated using human serum samples.

A colorimetric sensor based on multiple elements doped carbon dot nanozyme for rapid detection of 1-naphthol in human urine samples

The residual carbaryl in crops can cause serious damage to the human kidney and nervous system after entering the human body, which may be metabolized to 1-naphthol (1-NAP) and excreted through urine. 1-NAP is often used as the biomarker for carbaryl exposure, so the intake or leakage of carbaryl can be monitored by detecting the concentration of 1-NAP. Herein, Co, N, P ternary co-doped carbon dots (CoNP-CDs) derived from vitamin B12 were synthesized by a facile hydrothermal method. CoNP-CDs exhibited oxidase-like activity and excellent peroxidase-like activity, which was attributed to the Fenton-like reaction of Co2+/Co3+ and the presence of pyrrole N and P elements, which together provided multiple active sites for chromogenic substrates. Due to the dual enzyme-like activity of CoNP-CDs, hydroxyl radicals (OH) and superoxide radicals (O2−) were generated during the catalytic process, which could rapidly oxidize colorless 3,3′,5,5′-tetramethyl benzidine (TMB) to blue oxidation products (oxTMB). The α-carbon in 1-NAP can be attacked by OH, and the catalytic oxidation process of TMB can be inhibited by the consumption of OH, so that the blue color of the solution became lighter. Based on this principle, a smartphone-assisted colorimetric sensing platform was constructed for the detection of 1-NAP, and which resulted in a linear range of 1.07–37.3 μM and a visual detection limit of 0.68 μM. Moreover, the colorimetric sensing system showed satisfactory recoveries in the detection of human urine samples. The colorimetric sensing system owned the advantages of fast response, strong selectivity and simple operation, and provided a potential strategy for the on-site detection of 1-NAP.

Genomic analysis of Strongyloides stercoralis and Strongyloides fuelleborni in Bangladesh.

About 600 million people are estimated to be infected with Strongyloides stercoralis, the species that causes most of the human strongyloidiasis cases. S. stercoralis can also infect non-human primates (NHPs), dogs and cats, rendering these animals putative sources for zoonotic human S. stercoralis infection. S. fuelleborni is normally found in old world NHPs but occasionally also infects humans, mainly in Africa. Dogs in southeast Asia carry at least two types of Strongyloides, only one of which appears to be shared with humans ("dog only" and "human and dog" types). For S. stercoralis with molecular taxonomic information, there is a strong sampling bias towards southeast and east Asia and Australia. In order to extend the geographic range of sampling, we collected human and dog derived Strongyloides spp. and hookworms from two locations in Bangladesh and subjected them to molecular taxonomic and genomic analysis based on nuclear and mitochondrial sequences. All hookworms found were Necator americanus. Contrary to earlier studies in Asia, we noticed a rather high incidence of S. fuelleborni in humans. Also in this study, we found the two types of S. stercoralis and no indication for genetic isolation from the southeast Asian populations. However, we found one genomically "dog only" type S. stercoralis in a human sample and we found two worms in a dog sample that had a nuclear genome of the "dog only" but a mitochondrial genome of the "human and dog" type. S. fuelleborni may play a more prominent role as a human parasite in certain places in Asia than previously thought. The introgression of a mitochondria haplotype into the "dog only" population suggests that rare interbreeding between the two S. stercoralis types does occur and that exchange of genetic properties, for example a drug resistance, between the two types is conceivable.

Analysis of Aceclofenac, Ketorolac, and Sulindac in Human Urine Using the Microemulsion Electrokinetic Chromatography Method

A method to determine aceclofenac, ketorolac, and sulindac in human urine samples using microemulsion electrokinetic chromatography (MEEKC) has been developed in this study. The optimization of MEEKC conditions was carried out by changing the microemulsion compositions including the buffer pH, borate salt concentration, surfactant concentration, co-surfactant concentration, organic modifier concentration, and oil droplet concentration. The optimum separation of selected drugs was obtained with a composition of microemulsion containing 10 mM borate buffer pH 9, 0.5% sodium dodecyl sulphate (SDS), 6.6% n-butanol, 6.0% acetonitrile, and 0.8% ethyl acetate. Excellent linearity was obtained in the range concentration of 25 to 200 ppm with r2 &gt; 0.999. Limits of detection (LOD, S/N = 3) and limits of quantification (LOQ, S/N = 10) were 2.72 to 4.75 and 9.08 to 15.85 ppm, respectively. The solid-phase extraction (SPE) method using C-18 as an adsorbent and the solid phase micro-tip extraction (SPMTE) method using multiwalled carbon nanotubes (MWCNTs) as an adsorbent were used to clean-up and pre-concentrate the urine samples prior to the MEEKC analysis. The best recoveries of the selected drugs in the spiked urine sample were 91 to 103% with RSD of 1.26 to 4.03% (n = 3) using the SPE-MEEKC method.

Comparative study of two MIP-based electrochemical sensors for selective detection and quantification of the antiretroviral drug lopinavir in human serum

Thermal polymerization (TP) and electropolymerization (EP) are the two methods used in this study to explore the molecular imprinting process. To detect the antiviral medication lopinavir (LPV), an inhibitor of enzyme HIV-1 protease that is co-formulated with ritonavir (RTV) to extend its half-life in the body, with greater precision, these methods were merged with an electrochemical sensor. The sensors were created on glassy carbon electrodes (GCE) based on molecularly imprinted polymers (MIP) using TP with methacrylic acid (MAA) functional monomer and EP with p-aminobenzoic acid (PABA) functional monomer. Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), and electrochemical methods were utilized to examine the technical features of the suggested sensors. For both approaches, the necessary optimization investigations were carried out. Different LPV concentrations, ranging from 1.0 pM to 17.5 pM in drug solution and commercial human serum samples, were used to validate the analytical efficiency of the two sensors and compare their electroanalytical behaviour. For TP-LPV@MIP/GCE and EP-LPV@MIP/GCE, the corresponding limit of detection (LOD) was 2.68 × 10−13 M (0.169 pg mL−1) and 1.79 × 10−13 M (0.113 pg mL−1) in standard solutions, and 2.87 × 10−13 M (0.180 pg mL−1) and 2.91 × 10−13 M (0.183 pg mL−1) in serum samples. For the measurement of LPV in tablet form and serum samples, the proposed TP-LPV@MIP/GCE and EP-LPV@MIP/GCE sensors provide good recovery, demonstrating 99.85–101.16 % and 100.36–100.97 % recovery, respectively. The imprinting factor was utilized to demonstrate the selectivity of the suggested sensors by utilizing several anti-viral drugs that are structurally comparable to LPV. Additionally, the constructed sensors were examined for the potential impacts of interferences and the stability during the storage.

Inhibition of inflammatory osteoclasts accelerates callus remodeling in osteoporotic fractures by enhancing CGRP+TrkA+ signaling.

Impaired callus remodeling significantly contributes to the delayed healing of osteoporotic fractures; however, the underlying mechanisms remain unclear. Sensory neuronal signaling plays a crucial role in bone repair. In this study, we aimed to investigate the pathological mechanisms hindering bone remodeling in osteoporotic fractures, particularly focusing on the role of sensory neuronal signaling. We demonstrate that in ovariectomized (OVX) mice, the loss of CGRP+TrkA+ sensory neuronal signaling during callus remodeling correlates with increased Cx3cr1+iOCs expression within the bone callus. Conditional knockout of Cx3cr1+iOCs restored CGRP+TrkA+ sensory neuronal, enabling normal callus remodeling progression. Mechanistically, we further demonstrate that Cx3cr1+iOCs secrete Sema3A in the osteoporotic fracture repair microenvironment, inhibiting CGRP+TrkA+ sensory neurons' axonal regeneration and suppressing nerve-bone signaling exchange, thus hindering bone remodeling. Lastly, in human samples, we observed an association between the loss of CGRP+TrkA+ sensory neuronal signaling and increased expression of Cx3cr1+iOCs. In conclusion, enhancing CGRP+TrkA+ sensory nerve signaling by inhibiting Cx3cr1+iOCs activity presents a potential strategy for treating delayed healing in osteoporotic fractures. Inhibition of inflammatory osteoclasts enhances CGRP+TrkA+ signaling and accelerates callus remodeling in osteoporotic fractures.