Analytical biochemistry最新文献

Cyclic 3-phosphosphoglyceric anhydride (cPGA), a side product of glycolysis, acylates cellular amines and thiols to form amides and thioesters, respectively. Since these acylation reactions are harmful, organisms rely on a protein, known as DJ-1 in humans, to inactivate cPGA. Inactivation of cPGA likely plays a significant role in cytoprotection by DJ-1, but further progress in this direction is hampered by the lack of quantitative assays to measure the cPGA hydrolase activity of DJ-1 in biological samples. Here we report an optimized procedure for preparation of cPGA which is then used as a substrate to quantify enzymatic activity of DJ-1. The end-point assay for cPGA hydrolase uses dilute cell lysates to hydrolyze cPGA for 0.5–3.5 min followed by conversion of the remaining cPGA into thioester for spectrophotometric quantitation. We illustrate the utility of this assay by showing that higher levels of cPGA hydrolase activity result in better protection from acylation by cPGA. Moreover, the decrease of cPGA hydrolase activity due to oxidation of the catalytic cysteine of DJ-1 under oxidative stress and its subsequent recovery can be monitored using the assay. This relatively simple assay allows functional characterization of DJ-1 in biological samples through quantitative assessment of its cPGA hydrolase activity.

In recent years, with the in-depth study of circRNA, scholars have begun to discover a synergistic relationship between circRNA and microorganisms. Traditional wet lab experiments in biology require expensive financial, material, and human resources to investigate the relationship between circRNA and diseases. Therefore, we propose a new predictive model for inferring the association between circRNA and diseases, called HAGACDA. Specifically, we first aggregate the unique features of circRNA and diseases themselves through singular value decomposition, Pearson similarity, and the biological information characteristics of circRNA and diseases. Utilizing the competitive relationships between miRNA and other microorganisms, we construct a circRNA-miRNA-disease multi-source heterogeneous network. Subsequently, we use a relational graph attention network to aggregate features based on the structural connections between different nodes. To address the inherent limitations in capturing high-order patterns in edge sets, we integrate a hypergraph attention network to extract features of circRNA and diseases. Finally, association prediction scores for node pairs are obtained through a multilayer perceptron. We conducted a comprehensive analysis of the model, including comparative experiments and case studies. Experimental results demonstrate that our model accurately predicts the association between circRNA and diseases.

Xiaochaihu Decoction（XCHD）is a classic prescription for the treatment of fever, but the mechanism is not clear. In this study, We elucidated the mechanism of action through network pharmacology and molecular docking. A rat fever model was established to verify the prediction results of network pharmacology. The analysis revealed that 120 intersection targets existed between XCHD and fever. The TP53, STAT3, RELA, MAPK1, AKT1, TNF and MAPK14 as potential core targets of XCHD in fever treatment. GO and KEGG pathway enrichment analyses indicated that XCHD may act through pathways such as the AGE-RAGE signaling pathway in diabetic complications, TNF signaling pathway, IL-17 signaling pathway. Molecular docking results demonstrated that quercetin, kaempferol, β-sitosterol, stigmasterol and baicalein exhibited strong binding activity to key targets. Animal experiments showed that XCHD significantly reduced body temperature and levels of IL-1β, IL-6, TNF-α, NO, PGE2, and cAMP in rats with fever. Importantly, no significant difference was observed between the XCHD self-emulsifying nano phase plus suspension phase and XCHD group. XCHD exerts its therapeutic effects on fever through a multi-ingredient, multi-target, and multi-pathway approach.

CM310 is a recombinant humanized monoclonal antibody targeting Interleukin (IL)-4 receptor alpha (IL-4Rα). IL-4Rα blockade prevents IL-4 and IL-13 from binding to their receptor, thereby inhibiting downstream signaling pathways that drive Type 2 helper T-cell (Th2) inflammation. CM310 holds potential for treating Th2-related inflammatory diseases, such as asthma, atopic dermatitis and chronic sinusitis with nasal polyposis. In this study, a direct enzyme-linked immunosorbent assay (ELISA) was developed to measure the concentrations of CM310 in rat serum. Seven calibration standards (ranging from 25 to 1600 ng/mL) and three quality controls (70, 500 and 1250 ng/mL) were defined. The limit of detection (LOD), lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) were 13, 25 and 1600 ng/mL, respectively. The method exhibited excellent precision and accuracy and successfully applied to in vitro serum stability and pharmacokinetic (PK) studies. In conclusion, we have developed and validated a highly sensitive and selective method for measuring CM310 in Sprague-Dawley rats. The development and validation ELISA method met the acceptable criteria, which suggested that these can be applied to quantify CM310, as well as in PK studies.

In this work, we demonstrate the use of capillary electrophoresis and inductively coupled plasma mass spectrometry, as competitive methods primarily for ion chromatography, to determine changes in the concentration of small inorganic ions in the Nostoc sp. culture medium. Although macronutrients were analyzed by capillary electrophoresis with conductivity detection, micronutrients were analyzed by inductively coupled plasma mass spectrometry. The different light settings (light intensity and spectral composition) had a visible effect on the culture growth and depletion of calcium, magnesium, and phosphate ions, and iron and manganese elements when comparing the behavior under red or violet light with that under blue light.

As the main active glycoprotein of egg white, the biological functions of chicken ovomucin α- and β-subunit are closely related to the structure of glycans. However, the exact composition and structure of the subunit glycans are still unknown. We obtained highly pure chicken ovomucin α-subunit and β-subunit protein bands by the strategy combined with two-step isoelectric precipitation and SDS-PAGE gel electrophoresis. The ammonia-catalyzed one-pot procedure was then used to release and capture α-and β-subunit protein glycans with 1-phenyl- 3-Methyl-5-pyrazolone (PMP). The N/O-glycans of bis-PMP derivatives were purified and analyzed by LC-MS. More importantly, an effective dual modification was performed to accurately quantify neutral and sialylated O-glycans through methylamidation of sialic acid residues and simultaneously through carbonyl condensation reactions of reducing ends with PMP. We first showed that the α-subunit protein has only N-glycosylation modification, and the β-subunit only O-glycosylation, a total of 22 N-glycans and 20 O-glycans were identified in the α- and β-subunit, respectively. In addition, the complex N-glycan (47 %) and the sialylated O-glycan (77 %) are each major types of the above subunits. Such findings in this study provide a basis for studying the functional and biological activities of chicken ovomucin glycans.

When using High-field asymmetric ion mobility spectrometry (FAIMS) to process complex mixtures for deep learning analysis, there is a problem of poor recognition performance due to the lack of high-quality data and low sample diversity. In this paper, a Generative Adversarial Network (GAN) method is introduced to simulate and generate highly realistic and diverse spectral for expanding the dataset using real mixture spectral data of 15 classes collected by FAIMS. The mixed datasets were put into VGG and ResNeXt for testing respectively, and the experimental results proved that the best recognition effect was achieved when the ratio of real data to generated data was 1:4: where accuracy improved by 24.19 % and 6.43 %; precision improved by 23.71 % and 6.97 %; recall improved by 21.08 % and 7.09 %; and F1-score improved by 24.50 % and 8.23 %. The above results strongly demonstrate that GAN can effectively expand the data volume and increase the sample diversity without increasing the additional experimental cost, which significantly enhances the experimental effect of FAIMS spectral for the analysis of complex mixtures.

Chimeric-antigen-receptor-T (CAR-T) have heralded a paradigm shift in the landscape of cancer immunotherapy. Retrovirus-mediated gene transfer serves to deliver the specific CAR expressing cassette into T cells across a spectrum of basic research and clinical contests in cancer therapy. However, it is necessary to devise a precise and validated quantitative methodology tailored to the diverse CAR constructs. In the investigation, a TaqMan real-time qPCR method was developed, utilizing primers targeting ψ gene sequence. This method offers a swift, sensitive, reproducible, and accurate tool for evaluating retroviral copy numbers at the integrated DNA level. Importantly, the established qPCR exhibits no cross-reactivity with non-transduced T cells or tissues. The regression equation characterizing TaqMan real-time PCR dynamics is y = −3.3841x + 41.402 (R² = 0.999), showing an amplification efficiency of 97.47 %. Notably, the established qPCR method achieves a minimum detection of 43.1 copies/μL. Furthermore, both intra- and inter-group discrepancies remain below 4 %, underscoring the good repeatability of the established method. Our in vitro and in vivo results also support its sensitivity, specificity, and stability. Consequently, this method offers researchers with a cost-effective tool to quantify CAR copies both in vitro and in vivo.

GC-MS/MS combines the superior chromatographic resolution of GC with the specific and sensitive detection of tandem MS. On paper, it is an ideal system for the routine analyses of organic acids, yet very few studies have used and published such methods. This is likely due to several challenges highlighted in this communication. Briefly, the combination of EI ionization with MRM detection provides arguably insufficient specificity when targeting organic acids. Moreover, the narrow peaks generally produced by GC can lead to inaccurate quantification when the mass spectrometer's cycle time is too long. Potential solutions to these problems are discussed.

Nitro fatty acids (NO₂–FAs) are biologically active compounds produced from the reaction of unsaturated fatty acids with reactive nitrogen species (RNS). Due to their electrophilic nature, these endogenously produced metabolites can react with nucleophilic targets, producing a spectrum of modulatory and protective effects. Determination of NO₂–FAs in biological samples is challenging due to their low nanomolar to picomolar endogenous concentrations, indistinct metabolism, and distribution in many tissues and biofluids. Several attempts have been made to develop precise, standardized, and efficient methodologies for assessing physiological and pathophysiological processes to overcome the difficulties associated with their measurement. This review discusses those approaches utilizing liquid chromatography tandem mass spectrometry (LC‒MS/MS) and gas chromatography tandem mass spectrometry (GC‒MS/MS) for the quantification of NO₂–FAs, in addition to a summary of their laboratory synthesis and extraction from biological samples. Clinical associations with different pathological conditions, including hyperlipidaemia, cardiac ischemia and herpes simplex type 2 viral infection (HSV-2), are also discussed.