Analysis Of Serum Samples Research Articles

Untargeted Metabolomics and Liquid Biopsy Investigation of Circulating Biomarkers in Soft Tissue Sarcoma

Background: Soft tissue sarcomas (STSs) are rare, highly malignant mesenchymal tumours, comprising approximately 1% of all adult cancers and about 15% of paediatric solid tumours. STSs exhibit considerable genomic complexity with diverse subtypes, posing significant clinical challenges. Objectives: This study aims to characterise the molecular signature of primary STS through liquid biopsies and the untargeted metabolomic profiling of 75 patients, providing deep insights into cellular processes and potential therapeutic targets. Methods: This study analysed serum samples using nuclear magnetic resonance (NMR) spectroscopy for metabolomic profiling. Multivariate data analysis and machine learning classifiers were employed to identify biomarkers. Results: A panel of eleven significant deregulated metabolites were discovered in serum samples of patients with STS, with potential implications for cancer diagnosis and treatment. Conclusions: Choline decrease emerged as a marker for cancer progression, highlighting the potential of targeting its metabolism for therapeutic approaches in STS. The NMR analysis protocol proved effective for determining circulating biomarkers from liquid biopsies, making it suitable for rare disease research.

P1301 Bile acid composition and immunological effect in mouse model and IBD patients

Abstract Background Bile acids (BAs) were suggested to have immunomodulatory properties that could influence inflammatory pathways in Inflammatory Bowel Disease (IBD). Dysregulated BA metabolism has been observed in IBD patients, further presented in our study by an analysis of IBD patients BA profiles with association to gene expression and targeted treatment of organoid models to elucidate the specific mechanisms by which BAs may affect intestinal inflammation in IBD. Methods Small intestinal organoids were grown from WT mice and an IBD mouse model with caspase-8 KO in intestinal epithelial cells (IEC). Organoids treated with primary (1°) and secondary (2°) BAs (CDCA, LCA, DCA) and BA + JAK-inhibitor (CDCA + inhibitor) were analysed thoroughly by bulk-RNA sequencing to investigate effects of BA and possible strategies to ameliorate negative effects. BA composition of serum samples from healthy controls (HC) (n=25) and IBD patients (n= 85) was quantified using LC-MS/MS and compared to bulk-RNA sequencing results of the same individuals in order to identify correlations of BA abundancy and gene expression. Results In WT organoids, CDCA treatment induced a significant upregulation of numerous genes associated with key cellular processes such as immunological, cell death but also mitochondrial. In contrast, in genetically-predisposed IBD organoids predominantly displayed gene downregulation in response to CDCA, indicating a divergent transcriptional response between WT and KO genotypes. In IBD organoids, JAK inhibition in conjunction with CDCA significantly altered the expression profile, reversing the expression of several genes that were downregulated by CDCA alone. This reversal was gene-specific and suggests a compensatory or modulatory effect of JAK inhibition on CDCA signalling in a caspase-8 deficient background. Notably, this combinatorial effect was not observed in WT organoids, where CDCA + JAK inhibitor treatment did not substantially alter gene expression compared to CDCA alone. Targeted metabolomic analysis of patient serum samples revealed two distinct bile acid profiles within the IBD cohort. In one subgroup, serum was characterized by elevated levels of the 1°BA CDCA coupled with lower concentrations of both primary and conjugated 2°BAs. In contrast, a second subgroup displayed lower CDCA levels alongside increased concentrations of secondary bile acids. These bile acid composition patterns suggest differing metabolic states within the patient cohort. Conclusion Our findings reveal significant links between BA composition, gene expression, and JAK pathway modulation, offering new insights into BA–driven gene regulatory mechanisms in both murine organoids and an IBD patient cohort. References TRR241 IBDome Consortium, TRR 241 Research Initiative, Berlin-Erlangen, Germany

Concentrations of trace metals in serum: Implications for type 2 diabetes mellitus and gender difference

Trace metals play a pivotal role in maintaining normal and healthy physiology due to their various metabolic functions in humans. This study was performed to identify the association between trace metals and type 2 diabetes mellitus (T2DM) through a comparative analysis of serum samples from both healthy controls and T2DM patients. Additionally, we aim to explore the potential connection between gender differences and the concentration of trace metals in T2DM patients. The study included 103 participants, comprising 53 healthy controls and 50 T2DM patients, with blood samples stored at Eulji University Hospital (Daejeon, Korea). Serum concentrations of copper (Cu), zinc (Zn), manganese (Mn), and selenium (Se) and chromium (Cr) levels were measured and statistically analyzed by t-tests and logistic regression after the outliers replaced by mean. In the overall participants, serum concentrations of Cu and Mn in T2DM group was significantly different compared to the control group (P < 0.01). Logistic regression analysis indicated that Cu, Se and Mn concentration was significantly different in T2DM patients than in healthy controls in adjusted for age and sex. When examining trace metal concentrations by gender difference, Se were significantly lower in T2DM men (P < 0.01), while T2DM women exhibited significantly higher levels of Zn, Se and Mn (P < 0.05). Especially in women, logistic regression analysis indicated that Cu, Se and Mn concentration was significantly different in T2DM patients than in healthy control (adjusted for age OR of Cu, 0.92; 95 % CI 0.87 - 0.98, adjusted OR of Se, 3.04; 95 % CI, 1.71 - 5.34; adjusted OR of Mn, 4.50; 95 % CI, 2.09 - 9.68). This study showed that trace metal imbalances related to Se and Mn in patients with T2DM were particularly relevant in women. It highlights the significance of gender difference and suggests the potential benefits of regular monitoring of trace metal concentrations in the management of T2DM patients.

Postoperative Tongqi Formula ameliorates postoperative ileus via p38 MAPK signaling pathway and metabolic disorder.

This study investigated the mechanism by which the Postoperative Tongqi Formula (PTQF) treats postoperative ileus (POI) through regulation of the p38 MAPK signaling pathway, Zona occludens 1 (ZO-1) protein, and metabolism. The primary components of PTQF were characterized using UHPLC-Q-TOF-MS/MS. The identified compounds subsequently employed network pharmacology to predict the signaling pathways associated with the inflammatory phase of POI. The anti-inflammatory effects of PTQF were evaluated in vitro using RAW264.7cells. A rat model of POI was used to assess efficacy based on the spleen index and charcoal powder propulsion rat in the small intestine. Furthermore, pathological damage to the small intestine was analyzed using hematoxylin and eosin (HE) staining as well as immunofluorescence to evaluate ZO-1 protein expression. Inflammatory cytokine levels were quantified using enzyme-linked immunosorbent assay (ELISA). Subsequently, Western blot analysis was performed to examine the p38 MAPK signaling pathway. Finally, a metabolomics approach was employed to analyze serum samples to identify potential metabolic pathways. A total of 130 chemical constituents were identified in PTQF. Following the network pharmacology analysis of these compounds, the p38 MAPK signaling pathway was chosen for further investigation. In vitro, PTQF effectively inhibited inflammatory responses in RAW264.7cells. Results from the spleen index and charcoal powder propulsion rate indicated that PTQF alleviated the inflammatory phase of POI in rats by mitigating systemic and intestinal inflammation. This was supported by reduced levels of inflammatory factors, modulation of ZO-1 protein expression, and a decrease in p38 MAPK phosphorylation levels. Furthermore, serum metabolomics revealed nine differential metabolites linked to intestinal inflammation. PTQF mitigates inflammation and intestinal damage in POI rats by modulating inflammatory factors, ZO-1 protein expression, the p38 MAPK signaling pathway, and metabolic disturbances.

MicroRNA signatures of VO 2 peak in older adult participants of the Study of Muscle, Mobility and Aging.

Peak oxygen consumption during exercise (VO 2 peak), is a direct measure of cardiorespiratory fitness (CF), a key indicator of physical function and overall health. However, the molecular changes that underpin VO 2 peak variation are not clear. Our objective is to understand the miRNA signatures that relate to VO 2 peak variation, which could provide insights to novel mechanisms that contribute to low VO 2 peak. We used small RNA sequencing to analyze serum samples from 72 participants (70-79 yrs old, 53% female) of the Study of Muscle, Mobility and Aging (SOMMA). We analyzed samples from individuals with low or high VO 2 peak (N=18/group) as well as samples from 36 randomly selected participants spanning the entire spectrum of VO 2 peak. We used LIMMA analysis package for regression analysis and to identify differentially expressed miRNAs. We used receiver operating characteristic curve analysis to evaluate the Area Under the Curve (AUC) and sensitivity and specificity rates. We identified 1,055 miRNAs expressed in all serum samples. Expression of 65 miRNAs differed between participants with low and high VO 2 peak (p < 0.05). After p-value adjustment, expression of 5 miRNAs (miR-1301-3p, -431-5p, -501-5p, -519a-3p, and -18a-3p) remained significantly different (FDR = 0.05). The five miRNAs had AUC ranging from 0.77 to 0.84. The optimal sensitivity and specificity ranged from 70 to 80% and 80 to 90%, respectively. After adjustment for age and sex covariates, 46 miRNAs significantly correlated with VO 2 peak (p < 0.05) and miR-519a-3p remained significant based on adjusted of p-values. We identified a miRNA signature of VO 2 peak in older individuals that might provide insights to novel mechanisms that drive low VO 2 peak. Future studies will validate the findings in a larger, longitudinal study cohort.

Differential Diagnosis of Urinary Cancers by Surface-Enhanced Raman Spectroscopy and Machine Learning.

Bladder, kidney, and prostate cancers are prevalent urinary cancers, and developing efficient detection methods is of significance for the early diagnosis of them. However, noninvasive and sensitive detection of urinary cancers still challenges traditional techniques. In this study, we developed a SERS-based method to analyze serum samples from patients with urinary cancers. Rapid, label-free, and highly sensitive detection of human sera is achieved by cleaning and aggregating silver nanoparticles. Furthermore, a long short-term memory deep learning algorithm is used to distinguish serum spectra, and the performance of the model is evaluated by comparing the accuracy, sensitivity, specificity, and receiver operating characteristic curves. Taking advantage of SERS and machine learning in sensitivity and data processing, the three urinary cancers are clearly classified. This is the first attempt to exploit the SERS-machine learning strategy to discriminate multiple urinary cancers with clinical serum samples, and our results showed the potential application of this method in the early diagnosis and screening of cancers.

Multi-omics Analysis Reveals Key Gut Microbiota and Metabolites Closely Associated with Huntington's Disease.

Dysbiosis of the gut microbiota is closely associated with neurodegenerative diseases, including Huntington's disease (HD). Gut microbiome-derived metabolites are key factors in host-microbiome interactions. This study aimed to investigate the crucial gut microbiome and metabolites in HD and their correlations. Fecal and serum samples from 11 to 26 patients with HD, respectively, and 16 and 23 healthy controls, respectively, were collected. The fecal samples were used for shotgun metagenomics while the serum samples for metabolomics analysis. Integrated analysis of the metagenomics and metabolomics data was also conducted. Firmicutes, Bacteroidota, Proteobacteria, Uroviricota, Actinobacteria, and Verrucomicrobia were the dominant phyla. At the genus level, the presence of Bacteroides, Faecalibacterium, Parabacteroides, Alistipes, Dialister, and Christensenella was higher in HD patients, while the abundance of Lachnospira, Roseburia, Clostridium, Ruminococcus, Blautia, Butyricicoccus, Agathobaculum, Phocaeicola, Coprococcus, and Fusicatenibacter decreased. A total of 244 differential metabolites were identified and found to be enriched in the glycerophospholipid, nucleotide, biotin, galactose, and alpha-linolenic acid metabolic pathways. The AUC value from the integrated analysis (1) was higher than that from the analysis of the gut microbiota (0.8632). No significant differences were found in the ACE, Simpson, Shannon, Sobs, and Chao indexes between HD patients and controls. Our study determined crucial functional gut microbiota and potential biomarkers associated with HD pathogenesis, providing new insights into the role of the gut microbiota-brain axis in HD occurrence and development.

SERS profiling of blood serum filtrate components from patients with type II diabetes using 100 kDa filtration devices.

Blood carries some of the most valuable biomarkers for disease screening as it interacts with various tissues and organs in the body. Human blood serum is a reservoir of high molecular weight fraction (HMWF) and low molecular weight fraction (LMWF) proteins. The LMWF proteins are considered disease marker proteins and are often suppressed by HMWF proteins during analysis. This issue is addressed by using a filtration device to isolate the filtrate portion from blood serum samples having biomarker proteins up to the size of the cutoff value of the filtration device. In this research, 100 kDa filter devices are employed to obtain the filtrate portions from blood serum samples of type II diabetes mellitus patients and healthy volunteers, followed by characterization using surface-enhanced Raman spectroscopy (SERS) with silver nanoparticles (Ag NPs) as the SERS substrate. By using this approach, the collected filtrate is expected to contain marker proteins at a size of <100 kDa, which are associated with type II diabetes. These marker proteins are in a narrow size range (cutoff value of 100 kDa). Hence, they may be more easily identified by their characteristic SERS spectral features as compared to their analysis in the respective whole blood serum samples due to the exclusion of larger size proteins. These proteins that are present in the filtrate portions of type II diabetes may include adiponectin, C-reactive protein, insulin, leptin, RBP4, IL-6, TNF-α, Fibroblast Growth Factor 21 (FGF21), albumin, transthyretin, alpha-antitrypsin, transferrin, apolipoprotein A-1 (ApoA-1) and fetuin-A. Some prominent SERS bands are observed at 356 cm-1, 435 cm-1, 490 cm-1, 548 cm-1, 596 cm-1, 729 cm-1, 746 cm-1, 950 cm-1, 1330 cm-1, 1362 cm-1, 1573 cm-1 and 1689 cm-1, which differentiate type II diabetes patients from healthy individuals. Moreover, the SERS spectral data sets of various samples are classified using two chemometric approaches: principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA). The validation of the PLS-DA analysis classification model is indicated with 81% accuracy, 79% specificity, and 85% sensitivity, having a value of AUC = 0.75.

Serum Pharmacochemistry and Network Pharmacology Reveal Active Compounds and Mechanisms of the Huaxian Formula in Alleviating Radiation-Induced Pulmonary Fibrosis.

Radiation-induced pulmonary fibrosis (RIPF) is a serious complication of radiotherapy that lacks effective treatment options. The Huaxian formula (HXF), a traditional Chinese herbal remedy, shows promise in alleviating RIPF; however, its active ingredients and underlying mechanisms remain poorly understood. Through serum pharmacochemistry, network pharmacology, molecular docking, and experimental validation, we investigate the potential mechanisms of HXF in the prevention and treatment of radiation-induced pulmonary fibrosis (RIPF). Histological examination and non-invasive computed tomography (CT) scans in animal experiments revealed that HXF improved extracellular matrix collagen deposition in the lung tissue of irradiated mice and reduced fibrosis manifestations on CT images. Analysis of post-HXF administration serum samples identified 21 enriched compounds as potential active compounds, with 430 corresponding prospective targets. Overlapping these compounds with 991 RIPF-related genes yielded 127 genes primarily associated with the PI3K-Akt signaling pathway, EGFR tyrosine kinase inhibitor resistance, and the MAPK signaling pathway. Molecular docking indicated that key compounds in HXF serum, 5,7,8-trimethoxyflavone, and hyperoside, exhibited strong affinity with key targets. Finally, animal experiments confirmed that HXF significantly inhibited the expression of p-Akt and p-PI3K proteins in the lung tissue of irradiated mice. Our research results indicate that HXF may exert its effects on the prevention and treatment of radiation-induced pulmonary fibrosis (RIPF) through multiple pathways and targets, with the PI3K-Akt signaling pathway likely playing the most crucial role in this process.

Serum RNA profile reflects fluid status and atrophic retinal changes in neovascular age‐related macular degeneration

Aims/Purpose: The increasing prevalence of age‐related macular degeneration (AMD), a disease that can result in the loss of central vision, is an emerging problem worldwide due to aging societies. This creates a challenge for the healthcare system and understanding of the mechanisms of AMD pathogenesis will help in early, personalized, and efficient intervention. Current diagnostic methods rely on optical coherence tomography/angiography imaging, which identifies existing damages, but does not provide information on the mechanisms behind them. In this study, we analyzed mRNAs, lncRNA, and small ncRNAs profiles in the serum of nAMD patients and associated them with anatomical changes observed at the retinal level.Methods: RNA‐Seq analysis of human serum samples from neovascular AMD and control patients.Results: In the present work, we showed the difference in the serum RNA profile between neovascular AMD (nAMD) patients and controls. Moreover, the RNA profile of nAMD patients corresponded with anatomical changes in the retinal fluid compartments and atrophic changes and was affected by intravitreal anti‐vascular endothelial growth factor treatment.Conclusions: Therefore, serum could be used as a target for a liquid biopsy to explore anatomical changes in the eye during nAMD progression and treatment. This finding opens a new pathway in AMD studies, which are limited due to restricted access to live human target material and the limited value of animal models of human AMD.

A chiral porous organic cage–modified restricted-access material achieves online analysis of serum samples containing enantiomers and positional isomers

Restricted-access materials (RAMs) allow biological samples to directly enter the chromatographic column for analysis owing to the steric exclusion function ability for biomolecules and extraction function for small-molecule analytes, which promoting the development of rapid, efficient, and automated in vivo drug analysis. Few reports on chiral RAMs that have been used to analyze enantiomers and positional isomers in serum by direct injection in currently. In this study, a chiral porous organic cage material RCC3 was innovatively introduced into the inner surface of silica gel and modified the outer surface with polyethylene glycol to prepare a novel type of chiral RAM-RCC3, and reported the use of chiral RAM-RCC3 as a stationary phase for the separation of chiral compounds and positional isomers in blank serum using high-performance liquid chromatography. The novel RAM-RCC3 column exhibited good performance in the online analysis of nine enantiomers and five positional isomers in serum samples. The effects of analyte mass, temperature, and composition of the mobile phase on the separation of o-, m-, and p-nitrophenol in serum samples using the RAM-RCC3 column were also investigated. Even after 300 injections, the RAM-RCC3 column exhibited good reproducibility and stability. These results indicate the potential of the chiral RAM-RCC3 column as a stationary phase for direct injection analysis of both chiral separation and positional isomers in biological samples, which also rendering it suitable to be further developed as a new type of RAM for online analysis of various small molecules in biological samples.

Identification of serum biomarkers for cystic echinococcosis in sheep through untargeted metabolomic analysis using LC–MS/MS technology

Abstract Background Echinococcosis is a zoonotic disease caused by an Echinococcus tapeworm infection. While diagnostic methods for humans often rely on ultrasound imaging and immunodiagnostic techniques, diagnosis in intermediate hosts typically has no widely used diagnostic markers, hampering disease control efforts. Methods The differences in serum metabolites of sheep infected with Echinococcus granulosus and a control group were analyzed using ultrahigh-performance liquid chromatography (UHPLC) separation with tandem mass spectrometry (MS/MS) detection. This provided a basis for the early diagnosis and pathogenetic study of cystic echinococcosis (CE) in intermediate hosts at the metabolomics level. Orthogonal projections to latent structures–discriminant analysis (OPLS-DA) were used to analyze different metabolites in the serum of the two groups. The differentially abundant metabolites were entered into the MetaboAnalyst 5.0 online analysis website for processing, and the top-15-ranked metabolic pathways were set to produce bubble plots and differential abundance score plots, with a significant difference of P &lt; 0.05 and a false discovery rate (FDR) &lt; 0.1 as the screening conditions. Results Data analyses of serum samples from both groups identified a total of 1905 significantly different metabolites, where 841 metabolites were upregulated and 1064 metabolites were downregulated. Twelve metabolites were significantly upregulated and 21 metabolites were significantly downregulated in the experimental group. Then, the 1,7-dihydroxyxanthone, 2-methylbutyrylglycine, 3,3-dimethylglutaric acid, 5,12-dihydroxy-6,8,10,14,17-eicosapentaenoic acid, 9-hydroperoxy-10E,12Z,15Z-octadecatrienoic acid, and trimethylamine N-oxide 6 metabolites were selected as diagnostically valuable candidate biomarkers (area under the curve [AUC] &gt; 0.7). These differential metabolites are involved in various metabolic pathways, including amino acid metabolites (arginine, l-isoleucine, l-valine) and fatty acid metabolism (fenugreek, arachidonic acid, linolenic acid). Compared with the control group, sheep in the CE group had increased serum levels of fenugreek acid, while all other metabolites such as glycine showed significantly reduced serum levels (P &lt; 0.01). Conclusions Through non-targeted metabolomic analysis of the serum of CE-infected sheep, differential metabolites closely related to amino acid metabolism and the fatty acid metabolism pathway were identified. These differentially abundant metabolites can serve as biomarkers for diagnosing CE infection in intermediate sheep hosts. Graphical abstract

Comparative lipidomic and metabolomic profiling of mdx and severe mdx-apolipoprotein e-null mice

AbstractDespite its notoriously mild phenotype, the dystrophin-deficient mdx mouse is the most common model of Duchenne muscular dystrophy (DMD). By mimicking a human DMD-associated metabolic comorbidity, hyperlipidemia, in mdx mice by inactivating the apolipoprotein E gene (mdx-ApoE) we previously reported severe myofiber damage exacerbation via histology with large fibro-fatty infiltrates and phenotype humanization with ambulation dysfunction when fed a cholesterol- and triglyceride-rich Western diet (mdx-ApoEW). Herein, we performed comparative lipidomic and metabolomic analyses of muscle, liver and serum samples from mdx and mdx-ApoEW mice using solution and high-resolution-magic angle spinning (HR-MAS) 1H-NMR spectroscopy. Compared to mdx and regular chow-fed mdx-ApoE mice, we observed an order of magnitude increase in lipid deposition in gastrocnemius muscle of mdx-ApoEW mice including 11-fold elevations in -CH3 and -CH2 lipids, along with pronounced elevations in serum cholesterol, fatty acid, triglyceride and phospholipids. Hepatic lipids were also elevated but did not correlate with the extent of muscle lipid infiltration or differences in serum lipids. This study provides the first lipometabolomic signature of severe mdx lesions exacerbated by high circulating lipids and lends credence to claims that the liver, the main regulator of whole-body lipoprotein metabolism, may play only a minor role in this process.

Potential therapeutic effects of IL28RA inhibition on acute myocardial infarction through phosphorylated JAK1/STAT1 signaling pathways

While current coronary intervention therapies and surgical bypass procedures are widely utilized, the treatment of acute myocardial infarction (AMI) in the elderly continues to pose significant challenges. Following AMI, the body’s immune system is activated, resulting in the release of inflammatory mediators that exacerbate myocardial damage. Interleukin 28A (IL28A) and interleukin 28B (IL28B) may play a role in immune regulation post-AMI by specifically binding to interleukin 28 receptor alpha (IL28RA). However, the precise underlying mechanisms remain incompletely understood. This study aims to investigate the levels of IL28A and IL28B following AMI, as well as the protective effects of inhibiting IL28RA expression in the context of AMI and its potential mechanisms. We analyzed serum samples from 55 patients with AMI and 41 control individuals using ELISA to evaluate the levels of IL28A and IL28B, as well as to assess their correlation with the clinical parameters of the patients. Additionally, we established a mouse model of AMI and employed intramyocardial injection of lentivirus to knock down IL28RA in the myocardium. Echocardiography was utilized to compare structural and functional changes, while HE staining was conducted to analyze the infarct area and assess changes in myocardial tissue and cell morphology. The expressions of IL28A, IL28B, IL28RA, and JAK1/STAT1 pathway-related proteins in the infarct area were compared through immunofluorescence and Western blot analysis. Finally, TUNEL staining and the BAX/Bcl2 ratio were utilized to evaluate cardiomyocyte apoptosis. The study demonstrated that serum IL28A levels in patients with AMI were significantly elevated compared to those in normal controls, whereas IL28B levels were significantly reduced. Additionally, both IL28A and IL28B levels exhibit a linear relationship with high-density lipoprotein (HDL) and body mass index (BMI). In a mouse model, cardiac function deteriorated and ventricular structural changes were observed 14 days post-myocardial infarction relative to controls. The expressions of IL28A and IL28RA were significantly upregulated in the myocardium of the infarcted area, while IL28B levels showed no significant variation. Additionally, the ratios of p-JAK1/JAK1 and p-STAT1/STAT1 were significantly increased, accompanied by a notable rise in apoptotic cells within the myocardial infarction area. Importantly, the knockdown of IL28RA expression in the infarcted region effectively mitigated these alterations. These results suggest that IL28A but not IL28B contributes to the process post-AMI and may induce cardiomyocyte apoptosis through the JAK1/STAT1 pathway in conjunction with IL28RA.

Simultaneous isotyping and semi-quantitation of anti-drug antibodies to an IgG1 biotherapeutic using hybrid LBA-LC-MS/MS.

Commonly, ligand-binding platforms are being used for immunogenicity assessment, but with the recent advent of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for protein quantification, this technology has become an alternative for the measurement of anti-drug antibodies (ADAs), when combined with an immunocapture step to extract them out of the biological sample. The monoclonal antibody adalimumab was immobilized on magnetic beads to isolate ADAs against this drug from serum samples. Multiple repetitions of immunopurification were used to minimize nonspecific binding and improve drug tolerance while maintaining sufficient recovery. A subsequent tryptic digestion released peptides, from which unique peptide sequences, originating from the constant region of seven ADA subclasses, were selected. These were then analyzed by LC-MS/MS against (unextracted) subclass-specific reference standards for semi-quantification. With two immunocapture and two immunopurification steps, the method simultaneously measures the ADA subclasses IgG1, IgG2, IgG3, IgG4, IgM, IgE and IgA within their relevant ranges, with good repeatability and drug tolerance, and limited interference of endogenous immunoglobulins. The method was successfully applied for the analysis of serum samples of subjects dosed with adalimumab. Hybrid LBA-LC-MS/MS is a viable platform for measuring ADAs and adds value, especially when ADA isotyping is needed.

Sex-specific cytokine, chemokine, and growth factor signatures in T1D patients and progressors.

Numerous studies have reported altered cytokine levels in type 1 diabetes (T1D) patients, yet findings remain inconsistent. In this pilot study, we tested the hypothesis that circulating immune markers exhibit sex-based differences in T1D, both prior to and after disease onset. We analyzed 47-48 cytokine, chemokine, and growth factor levels in two cohorts. To assess post-disease differences, we analyzed serum samples from 25 controls and 25 T1D patients. To examine pre-disease progression, we utilized samples from 21 control children and 16 T1D progressors, collected at age 5 years before disease onset. Across all T1D patients and controls, only macrophage colony-stimulating factor and interleukin (IL)-6 showed significant differences. However, we identified notable alterations when comparing sex-age-matched controls and T1D samples. Female T1D patients exhibited lower levels of inflammatory cytokines (tumor necrosis factor-α, IL-6, IL-1a), Th2 cytokines (IL-4, IL-13), and chemokines (macrophage inflammatory protein (MIP)-1α, regulated upon activation, normal T cell expressed and secreted, MIP-3) compared to female controls, differences that were not observed in males. Notably, IL-22 was lower in female T1D patients compared to female controls, whereas it was higher in male T1D patients compared to male controls. Male T1D patients showed elevated levels of growth factors (epidermal growth factor, platelet-derived growth factor-AB/BB) compared to male controls. In T1D progressors, growth-regulated alpha was lower compared to controls in both sexes. Multiple regression analysis further revealed associations between cytokine levels and factors such as age, BMI, and breastfeeding duration. Overall, our findings serve as a proof of concept, highlighting the importance of sex-specific differences in T1D pathogenesis. However, follow-up studies with larger sample sizes are needed to validate and generalize these results.

Longitudinal analysis of serum intestinal fatty acid-binding protein in a patient with non-occlusive mesenteric ischemia following brachial plexus block-induced hypotension: a case study.

Intestinal fatty acid-binding protein (I-FABP) is a promising biomarker for small-bowel ischemia including non-occlusive mesenteric ischemia (NOMI). A 75-year-old woman with diabetic nephropathy sustained a distal radius fracture. Two days later, she underwent a brachial plexus block to facilitate orthopedic surgery, which resulted in hypotension. Despite prompt fluid resuscitation and ephedrine administration, the patient developed abdominal pain. Contrast-enhanced computed tomography revealed hepatic portal venous gas, but no direct evidence of small-bowel ischemia. The gastrointestinal surgery team opted for cautious in-hospital observation overnight. Unfortunately, the patient's condition significantly worsened the following day, prompting an urgent laparotomy. Surgery confirmed ileal segment necrosis, macroscopically characterized by a distinctive geographic pattern. Retrospective analysis of stored serum samples using a human enzyme-linked immunosorbent assay demonstrated that I-FABP levels were moderately elevated (7.2ng/mL) at the initial outpatient visit for the fracture, peaked (17.9ng/mL) at the clinical onset of NOMI, and returned to normal (0.7ng/mL) by postoperative day 2. Serum I-FABP levels correlated with the progression of NOMI, showing potential as an early detection marker. However, the longitudinal analysis of serum I-FABP also highlighted significant challenges of this biomarker, including the influence of renal function and the importance of sampling timing.

An isolate of human blood-associated partitivirus naturally infects the phytopathogenic fungus Bipolaris maydis.

Human blood-associated partitivirus (HuBPV) was first identified through metagenomic analysis of serum samples from two Peruvians, but its natural host remains unknown. Here, we report the detection of an HuBPV strain (HuBPV-Bm) in the phytopathogenic fungus Bipolaris maydis strain HN11 in Hubei Province, China. The dsRNA1 and dsRNA2 of HuBPV-Bm show more than 97.6% and 98.8% nucleotide sequence identity, respectively, to those from the metagenomically discovered HuBPV strain (HuBPV-M). Notably, HuBPV-Bm contains a third dsRNA segment that was not reported for HuBPV-M. All mycelia derived from individual asexual spores of HN11 tested positive for HuBPV-Bm, as did nine out of 293 B. maydis strains collected across Hubei.

The Preventive Effect of Zinc Sulfate against Olanzapine-Induced Testicular Toxicity in Male Rats.

Male infertility is a complex and multifactorial clinical condition affecting a large population attributed to several factors, including perturbation in oxidative stress and the level of essential trace elements. Oxidative stress exerts multiple issues related to reproductive health, including male infertility, decreased sperm motility, sperm DNA damage, and an increased susceptibility to genetic disorders. Besides chemical toxins and food allergens in junk food items, many drugs can also lead to male infertility. Olanzapine (OLZ), a general antipsychotic drug, has also been reported to induce male fertility. A great deal of literature entails that supplementation of zinc can alleviate oxidative stress-related clinical complications, including male infertility. This study investigates the potential protective effects of zinc sulfate (ZnSO4) on OLZ-induced male infertility. In the present study, there were four treatment groups (n = 6): group 1 as control (without any treatment); group 2 treated with OLZ (10mg/kg) orally daily for 6weeks; groups 3 and 4 treated with 50mg/kg and 100mg/kg of ZnSO4 respectively in pre-exposed OLZ (10mg/kg) orally daily for 6weeks. After completion of the treatment, the biochemical analysis of serum and tissue samples demonstrated that group 2 had compromised levels of antioxidant parameters (superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH)) as well as elevated levels of stress parameters (oxidized glutathione (GSSG), malondialdehyde (MDA), and nitric oxide (NO)) as compared to the control by a significant extent (p ≤ 0.05). However, supplementation of ZnSO4 significantly corrected all these parameters in a dose-dependent way in groups 3 and 4 (p ≤ 0.05). Other health indicators, like taurine, enzyme Q10, phosphatidylcholine, ascorbic acid, and vitamin E, were also observed to improve prominently with the supplementation of ZnSO4. Intriguingly, all the fertility parameters (sperm motility, count and level of testosterone) were also found to be significantly enhanced with decreased sperm abnormalities in both the combination groups. The histological evaluation of the testis section also agreed with the biochemical analysis in this investigation. Therefore, the study strongly advocates that supplementation of Zn can ameliorate OLZ-induced male infertility to a significant extent.

Highly Sensitive Multiplexed Sensing of miRNAs in a Gastric Cancer Patient's Liquid Biopsy.

Gastric cancer (GC) is one of the leading causes of cancer mortality in the world. Most patients are in the advanced stage of the disease at the time of diagnosis because the symptoms of early gastric cancer patients are not obvious. Early diagnosis of gastric cancer is still challenging due to the high cost, invasiveness, and low accuracy of traditional diagnostic methods such as endoscopy and biopsy. Herein, we develop clinically accurate and highly sensitive detection of multiple GC miRNA biomarkers in human serum using an isothermal nucleic acid primer exchange reaction (PER). The isothermal nucleic acid primer exchange reaction demonstrates high sensitivity and robustness, exemplified by a one-pot reaction achieving a detection limit of 28.71 fM. By quantifying the levels of three miRNA biomarkers selected through bioinformatics analysis in gastric cancer serum samples, the diagnostic approach effectively distinguished between clinical gastric cancer patients (n = 25) and noncancer controls (n = 10). The performance of our three-miRNA signature in discriminating between GC and controls was as follows: area under the curve (AUC): 0.808, sensitivity: 89%, specificity: 88%, positive predictive value (PPV): 96%, and negative predictive value (NPV): 70%.