Background Sensitive detection of protein biomarkers in neural tissue is important for both research and diagnostic applications. Enzyme-linked immunosorbent assay (ELISA) is a gold-standard immunoassay known for high sensitivity and quantification, whereas colloidal gold lateral flow assays offer rapid, instrument-free testing but are generally qualitative. This study compared ELISA and colloidal gold strip tests for detecting acetylcholinesterase (AChE) in formalin-fixed cadaveric nerve tissues. Five cadavers (3 males, 2 females) without neurological disease or limb trauma provided proximal and distal nerve segments for analysis. Tissues were formalin-fixed, heat-treated to reverse cross-links, and extracted for protein. A bicinchoninic acid (BCA) assay measured total protein yields, and an ELISA quantified AChE concentration. In parallel, a colloidal gold immunochromatographic strip test was applied to diluted extracts to visually detect AChE. Methods We evaluated detection sensitivity (limit of detection and positive detection rate), reproducibility (intra-assay variability), and quantitative agreement between methods. Results ELISA detected AChE in 12/12 extracted nerve samples, with concentrations ranging from ~0.2 to 66 ng/mL in tissue extract (mean ~15 ng/mL). The colloidal gold strips, by contrast, returned visible positive lines only for samples above ~10–20 ng/mL AChE; low-level samples yielded no signal. ELISA showed a lower limit of detection around 0.5 ng/mL, approximately one order of magnitude lower than the strip test. ELISA measurements were highly reproducible (the duplicate-well coefficient of variation ~10–15%), whereas the lateral flow results were more variable near the cutoff and required subjective interpretation of faint test lines. A strong rank correlation ( ρ ≈ 0.9) was found between ELISA concentrations and strip test positivity thresholds. However, Bland–Altman analysis revealed the strip method systematically under-reported AChE levels, highlighting poor quantitative agreement. Conclusion The ELISA demonstrated superior sensitivity and accuracy for AChE in formalin-fixed neural tissue extracts, detecting low concentrations that the colloidal gold lateral flow assay missed. While the rapid strip test may be useful for quick yes/no identification of high-abundance biomarkers in nerve samples, it lacks the sensitivity and quantitative precision necessary for reliable neural protein diagnostics. Integration of more sensitive detection labels or reader devices would be required to bridge the performance gap between lateral flow assays and ELISA in this context.

Free flavin adenine dinucleotide (FAD) is metabolized to flavin mononucleotide (FMN) and adenine monophosphate (AMP) by hydrolases and to 4',5'-cyclic phosphoriboflavin (cFMN) and AMP by the triose kinase FMN cyclase (TKFC). Yet, the lack of analytical standards for cFMN might have resulted in the incidence of cFMN in biological specimens being underreported. To address this shortcoming, cFMN was synthesized from either FMN or FAD. The optimization of the FAD to cFMN reaction conditions revealed that an equimolar ratio of ZnSO4 and FAD yielded pure cFMN upon the precipitation of AMP-Zn salts. cFMN is stable to aqueous acidic and basic conditions and is readily extracted from biological samples for detection by liquid chromatography coupled with mass spectrometry. Although cFMN is hydrolyzed by liver tissue extracts to FMN and riboflavin, the mechanisms for this conversion remain elusive.

Advances in oncological and haematological treatments have markedly improved childhood survival, yet gonadotoxic therapies often compromise testicular function. Testicular tissue extraction (TTE) with cryopreservation is increasingly offered to prepubertal boys at high risk of infertility; however, data on long-term endocrine and fertility outcomes remain limited. This study evaluated the long-term endocrine and exocrine testicular function of males who underwent TTE prior to gonadotoxic therapy. This was a retrospective, observational, single-centre study including 50 males treated between 2009 and 2022 at Lille University Hospital (median age at TTE: 5.6 years; median age at last follow-up: 14.6 years). Underlying conditions comprised malignant haematological disorders (52%), non-malignant diseases (34%), and solid tumours (14%); 80% underwent allogeneic haematopoietic stem cell transplantation. Serial clinical assessments and biochemical markers of Leydig and Sertoli cell function (LH, FSH, testosterone, inhibin B, AMH) were recorded from puberty onset to young adulthood. Data were stratified by treatment intensity, pubertal timing, and disease type. Post-pubertal semen analysis was performed in consenting individuals. All participants experienced spontaneous pubertal onset. Sertoli cell dysfunction, evidenced by elevated FSH and persistently low inhibin B, was detectable early in puberty and persisted into adulthood. At Tanner stage 5, 18% had elevated LH, 75% elevated FSH, and 89% low inhibin B; comparable rates were observed in adulthood. Radiotherapy was associated with higher gonadotropin levels at equivalent chemotherapy doses, and Sertoli cell impairment was more pronounced in malignant disease and peri-pubertal treatment. Among 12 semen analyses, spermatozoa were detected in 42%, all in non-malignant cases without radiotherapy. Males undergoing TTE prior to gonadotoxic therapy frequently develop persistent Sertoli cell dysfunction, with limited fertility potential in many cases. These findings underscore the importance of systematic, long-term andrological surveillance and personalised fertility counselling in this high-risk population.

MED12 exon 2 mutation is the most frequent mutation associated with uterine leiomyomas. MED12 wild-type leiomyomas have a higher growth potential than mutant leiomyomas, suggesting that the mutation limits leiomyoma growth. MED12 forms a complex with CDK8 and is involved in the phosphorylation of RNA polymerase II, playing a role in transcriptional regulation. However, its mechanism of action in leiomyoma growth is not clear. We aimed to clarify the relationship between MED12 mutation status, response to gonadotropin-releasing hormone (GnRH) agonist treatment, and CDK8 activity in leiomyomas. We also examined the effects of CDK8 inhibitors on primary cultured uterine leiomyoma cells. We classified 44 surgically removed uterine leiomyomas into four groups according to GnRH agonist use and MED12 mutation status. CDK8 was co-immunoprecipitated from leiomyoma tissue extracts using MED12 antibody to test its kinase activity in vitro, and the amount of phosphorylated substrate was measured. Cell proliferation and apoptosis of primary cultured MED12 wild-type leiomyoma cells were evaluated in the presence of a CDK8 inhibitor and sex steroid hormones. Of the 44 leiomyomas tested, 11 MED12 wild-type leiomyomas without preoperative GnRH agonist treatment had significantly higher CDK8 activity than nine GnRH agonist-treated MED12 wild-type leiomyomas and 15 leiomyomas with MED12 mutations without GnRH agonist treatment. Treatment of primary cultured MED12 wild-type cells with CDK8 inhibitors significantly inhibited cell growth and increased apoptosis. MED12 wild-type leiomyoma cells without GnRH agonist treatment showed high CDK8 activity, and inhibition of CDK8 activity suppressed cell growth in vitro.

Glucocorticoids are produced through activation of the hypothalamic-pituitary-adrenal (HPA) axis, initiated by the release of corticotropin-releasing factor (CRF) from the hypothalamus. CRF acts through two receptor subtypes, CRF1 and CRF2. However, the specific contributions of CRF1 and CRF2 receptors to age-related changes in brain glucocorticoid activity remain largely unexplored. In certain tissues, including the hippocampus, glucocorticoid signaling is further amplified by the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which regenerates inactive glucocorticoid metabolites into their active form. Notably, prior research investigating the role of hippocampal 11β-HSD1 in aging has focused exclusively on male subjects. In this study, we used genetic mouse models lacking functional CRF1 or CRF2 receptors to investigate their respective roles in regulating hippocampal 11β-HSD1 activity and glucocorticoid levels across age and sex. Mice of both sexes at 6 and 18 months of age were analyzed. Hippocampal 11β-HSD1 activity was assessed by measuring the ratio of corticosterone to dehydrocorticosterone using mass spectrometry in tissue extracts from CRF1 and CRF2 wild-type (WT), heterozygous (HET), and knockout (KO) mice. Our results demonstrate that hippocampal 11β-HSD1 activity increases with age in female CRF1 WT and HET mice but not in CRF1 KO females. In contrast, aged males exhibit elevated 11β-HSD1 activity regardless of CRF1 genotype. In CRF1 males, the age-related increase in hippocampal 11β-HSD1 activity is associated with higher hippocampal corticosterone levels, whereas in CRF1 females, it corresponds with a decrease in hippocampal dehydrocorticosterone. CRF1 deficiency leads to reduced hippocampal levels of both corticosterone and dehydrocorticosterone in males and females at both ages. CRF1 deficiency is also associated with decreased plasma corticosterone levels in both male and female mice. Male, but not female, CRF2 mice show an age-dependent increase in hippocampal 11β-HSD1 activity, which is not altered by CRF2 deficiency. Moreover, CRF2 deficiency results in increased plasma corticosterone in female, but not in male, mice. Overall, our findings reveal that hippocampal 11β-HSD1 activity increases with age in both sexes. In females, this increase is dependent on the presence of functional CRF1 receptors. In contrast, males exhibit age-related increases in 11β-HSD1 activity independent of CRF1 function. These findings underscore the importance of considering sex as a biological variable when developing therapeutic strategies targeting 11β-HSD1 to mitigate age-related memory decline.

ABSTRACT Vibrio cholerae (V. cholerae) is a type of bacterium that causes cholera, a severe diarrheal disease globally affecting hundreds of people annually. However, the effect of the V. cholerae toxin on oyster metabolite signatures has not been well studied. In this study, nuclear magnetic resonance (NMR) based metabolomics was applied to investigate the metabolic level response of eastern oysters (Crassostrea virginica) to cholera toxin (CT), under low concentrations. Our study demonstrated that the decrease of branched-chain amino acids (BCAAs) in oysters was a response to CT exposure at low concentrations (10 ng/mL) in gill and mantle extracts. Metabolites such as leucine and isoleucine were significantly decreased in gills with toxin exposure at 10 ng/mL, and similar but weaker changes were also observed at 1 ng/mL, indicating an early response to CT. However, the trend reversed at 20 ng/mL, with acetate and propionate significantly increased over control (p < 0.07), which is a sign of antioxidant defenses that could help the recovery of the BCAAs. In the hemolymph study, acetate and propionate levels correlated strongly with those in the tissue extracts at 20 ng/mL, suggesting that hemolymph metabolites begin contributing to gill metabolic perturbations. More importantly, a principal component analysis (PCA) also revealed a partial separation between the control and the 20 ng/mL CT group, indicating potential major perturbations in hemolymph metabolites. This study provides evidence that metabolites in oyster tissues resulting from exposure to Vibrio toxin can serve as a new early warning system for predicting potential human pathogen risks in both environmental and seafood exposure.

Ligation-mediated circuit-driven cascade amplification with high utilization rate of template for lncRNA MALAT1 detection in cancer mice tissues.

Environmental DNA (eDNA) analysis enables biodiversity monitoring by detecting organisms from trace genetic material, but high reagent costs, cold-chain logistics and computational demands limit its broader use, particularly in resource-limited settings. To address these challenges and improve accessibility, we directly compared multiple workflow components, including four DNA extraction methods, two primer sets, three Nanopore basecalling models, and two demultiplexing pipelines. Across 48 workflow combinations tested in an aquarium with 15 fish species, we mapped trade-offs between cost, sensitivity, and processing speed to assess where time and resource savings are possible without compromising detection. Workflows using the Qiagen Blood and Tissue (BT) extraction kit and amplification using the MiFish-U primer set provided the highest sensitivity, detecting ≥ 12 of 15 species by ~3-5 h and reaching the 15-OTU plateau at ~8-10 h with Oxford Nanopore's high accuracy (HAC) basecalling model. Chelex, an alternative lower-cost extraction method, showed partial recovery only (≤ 9 OTUs by 61 h) even with extended sequencing, and did not recover all 15 OTUs. DirectPCR and QuickExtract offered field-friendly extraction alternatives that achieved comparable recovery in ~10-12 h, though their cost-effectiveness varied. While the MarVer1 primer was designed to broaden vertebrate detection, it recovered the same fish species as MiFish-U, though with fewer total reads. Real-time sequencing trials (0-61 h) revealed that high-efficiency workflows (BT + HAC) reached detection plateaus rapidly, indicating sequencing time can be reduced without sacrificing accuracy. The OBITools4 bioinformatics pipeline enabled automated demultiplexing but discarded more reads than an alternative, ONTbarcoder2.3, which retained low-abundance taxa at the cost of manual curation. Rather than identifying a single 'best' workflow, this study provides a transparent decision framework for prioritising cost, speed, and sensitivity in eDNA applications, supporting scalable, cost-effective eDNA monitoring in resource-limited settings.

Calprotectin (S100A8/A9) constitutes approximately 60% of the cytosolic protein content of neutrophils and serves as a marker of leukocyte activation and migration, providing valuable insight into the intensity and pattern of inflammation. This study aimed to evaluate the tissue expression and quantification of calprotectin in anorectal samples from patients with inflammatory bowel disease (IBD) using immunochemiluminescence and immunohistochemistry. Anti-calprotectin antibodies were conjugated with acridine ester for chemiluminescent detection and applied to tissue extracts. In parallel, indirect immunohistochemistry was performed on paraffin-embedded anorectal sections from patients with Crohn’s disease and ulcerative colitis, and from non-IBD controls. Quantitative and semiquantitative analyses were compared between groups. Calprotectin levels were significantly elevated in IBD tissues compared with controls (p &lt; 0.05) in both detection methods. The chemiluminescent assay demonstrated higher analytical sensitivity, enabling quantification even in samples with mild histological inflammation. Increased tissue calprotectin reflects enhanced neutrophil infiltration and activation within the intestinal mucosa, corroborating its role as a local biomarker of inflammatory activity in IBD.

General background: Aromatic herbs from the Lamiaceae family represent valuable sources of rosmarinic acid (RA), a polyphenolic compound with notable antioxidant, anti-inflammatory, antimicrobial, and neuroprotective properties. Specific background: Traditional extraction methods such as Soxhlet and maceration consume excessive solvents and time while potentially degrading thermolabile compounds. Knowledge gap: Despite growing interest in green extraction technologies, limited validated protocols exist for combining ultrasound-assisted extraction (UAE) with high-performance liquid chromatography (HPLC) for efficient RA quantification across multiple aromatic species. Aims: This study developed and validated a sustainable UAE-HPLC method for extracting and quantifying RA from six aromatic herbs: Laurus nobilis, Thymus vulgaris, Salvia rosmarinus, Anethum graveolens, Mentha viridis, and Salvia officinalis. Results: Using ethanol:water (70:30 v/v) at 40 kHz and 250 W, optimal extraction occurred within 2.5-10 minutes, with Thymus vulgaris yielding the highest RA concentration (170,500 µg/mL). HPLC analysis demonstrated excellent linearity (R² &gt; 0.998) and reproducibility. Novelty: This approach significantly reduces solvent consumption and extraction time while maintaining analytical precision. Implications: The validated UAE-HPLC platform provides a robust, environmentally friendly analytical framework for sustainable utilization of RA-rich herbs in nutraceutical and pharmaceutical applications.Highlight : UAE offers a rapid, low-solvent, and environmentally friendly approach for extracting RA. Variation in RA yields reflects differences in plant tissue structure and extraction conditions. HPLC provides reliable quantification, supporting accurate and consistent analysis of RA. Keywords : Rosmarinic Acid, Aromatic Herbs, Ultrasound-assisted Extraction, HPLC, Green Extraction

A hypothesis that has gained traction in Alzheimer's disease (AD) research is the autoimmune hypothesis, which posits that autoantibodies generated in the central nervous system attack neurons, leading to AD manifestations. To uncover such autoantibodies against neuronal proteins in AD, reliable, sensitive and specific analytical methods need to be developed. Our method for the unbiased autoantibody identification in cerebrospinal fluid (CSF) includes the following steps: (1) capture of G-class immunoglobulins from 10uL of CSF using protein G magnetic beads, (2) exposure to a brain tissue extract, leading to cognate antigen capture, (3) reduction by dithiothreitol and digestion with trypsin, (4) tryptic peptides extraction and subjection to analysis by mass spectrometry (MS) to identify the bound proteins. For method optimization, we used CSF spiked with known amounts of a monoclonal antibody against Kallikrein 6 (KLK6ab), which is abundant in normal CSF. MS was performed with two machines coupled to liquid chromatography: (a) an Orbitrap Astral MS operating in data-independent acquisition (DIA) mode and 20min gradient, (b) a Bruker timsTOF Pro 2, operating in DIA mode and at either 22min or 44min gradient. Interpretation was done with Spectronaut. We resolved the CSF proteome (20uL) with a shotgun approach and identified 1988.1 proteins with machine (a) in DIA (1/5 sample) and 1241.3 with (b) in DIA. We resolved the KLK6 capture, finding a protein intensity (PG.Quantity) of 1117.01 in (a), and a protein intensity of 510.68 in (b) after adding 1ng of KLK6ab in both samples. The limit of detection for both machines (a) and (b) was 0.2ng of KLK6ab. Previous analyses also revealed that with a DDA analysis, the limit of detection was 5ng of KLK6ab. We optimized our method for identification of endogenous CSF autoantibodies on various mass spectrometric platforms using an internal antibody control that assures optimal performance of all experimental runs. We believe that this assay will be instrumental in identifying novel autoantibodies that may be related to AD pathogenesis.

Obesity promotes a range of associated conditions, including hearing impairment; however, mechanisms are lacking. Self-evidently, an insult on any cellular constituent of the auditory organ can disrupt hearing. Here, using the mouse auditory cell line, HEI-OC1, we provide insights into adipose-associated ototoxicity. Adipose extracts from mice with obesity, diet- or genetically induced, suppress HEI-OC1's survival and ATP generation. Proteomic profiling shows an upregulation of the inflammatory response pathway and proteins such as Podoplanin and Low-density lipoprotein receptor. Likewise, the Programmed cell death 4 (PDCD4) protein was induced. These results correspond to a downregulation of glycolysis and oxidative phosphorylation but an upregulation of the G2/M checkpoint. Additionally, pathways such as IL6-JAK-STAT3, IL2-STAT5, interferon gamma response, cholesterol response, bile acid metabolism, RAS, Apoptosis, and TGF-β were upregulated. Furthermore, the adipose extracts cause cellular morphological changes consistent with cells under stress. Functional assays point to alterations in levels of proteins related to calcium and ER homeostasis/stress. The ER-resident protein SARAF, an inhibitor of calcium overfilling, is among the proteins markedly downregulated. GRP78 protein levels increased, suggesting ER/calcium stress. Finally, Thapsigargin impairs HEI-OC1 survival, reminiscent of the effect of the adipose tissue extracts. Our analyses warrant further exploration of inflammation and ER/calcium stress in connection to obesity-associated ototoxicity.

Ratio of placental and umbilical cord Vascular Endothelial Growth Factor (VEGF) levels in pregnancies with preeclampsia and its association with maternal variables and fetal outcome.

Background and Purpose Although the impact of nicotine on the dopaminergic system is well established, its effects on neural activity in the brain regions implicated in addiction remain unclear. The major objective of the study was to assess the impact of acute nicotine on neuronal and astrocytic metabolic activity in the prefrontal cortex, cerebral cortex and hippocampus of awake mice. Experimental Approach Nicotine (0.0125–2.00 mg kg −1 ) was administered subcutaneously to 2‐ to 2.5‐month‐old C57BL/6NCrl male mice. The neuronal and astrocytic metabolic activity was measured by infusing [1,6‐ 13 C 2 ]glucose and [2‐ 13 C]acetate, respectively, 15 min after injection, and monitoring amino acids labelling in the 1 H‐[ 13 C]‐NMR spectrum of brain tissue extracts. Key Results Nicotine perturbed glucose metabolism in a dose‐ and brain‐ region‐dependent manner. At lower doses, it enhanced the rate of glucose oxidation in glutamatergic neurons in the hippocampus (0.0125 mg kg −1 ) and prefrontal cortex (0.025 mg kg −1 ), with no change in the cerebral cortex. In contrast, a higher nicotine dose (1.0 mg kg −1 ) suppressed glutamatergic and GABAergic neurometabolic activity in all three brain regions. Nicotine did not affect the astrocytic metabolic activity at the lower dose (0.025 mg kg −1 ) but suppressed it at the high dose (2.0 mg kg −1 ). Conclusions and Implications Nicotine has biphasic impacts on glutamatergic activity, enhancing excitatory activity at low doses but reducing both excitatory and inhibitory activity at higher doses. Most interestingly, acute nicotine increases neuronal excitability by shifting the excitation‐to‐inhibition balance in the prefrontal cortex, a critical component of the mesocortical circuitry.

Flax from cultivar Alexin was grown on three different water regimes: non-irrigated, half dose and full-dose irrigated. Seeds were harvested, extracted in 70 % ethanol solution and analyzed for antioxidant activity, using the DPPH method.Results show an extreme variation of antioxidant activity with irrigation regime, from just 3.18 % in non-irrigated flax, to 48.24 % in full-dose irrigated one, with 18.08 % at half dose irrigation, corresponding to IC50 values of 45.68–106.88. Irrigation, even with half dose raised linseed tissue extract potential to strongly antioxidant and to very strongly antioxidant at full dose..These results can be linked to data showing a major increase in concentrations of some key compounds, especially of phenolic compounds.

Colorectal cancer (CRC) remains a significant global health burden, mainly due to late diagnosis and chemotherapy resistance. Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with tumor progression, has emerged as a promising biomarker in CRC. However, its clinical utility is limited by the lack of rapid and accessible detection methods. In this study, we report an electrochemical immunotechnology for the sensitive and selective quantification of MIF protein in CRC tissue samples. By combining magnetic microparticles (MMPs), antibody-based recognition, horseradish peroxidase (HRP) labeling, and amperometric transduction at disposable screen-printed carbon electrodes (SPCEs), the developed methodology displayed a linear dynamic range from 0.24 to 20 ng mL−1, enabling quantification across clinically relevant MIF levels, and achieving a low limit of detection (0.07 ng mL−1). In addition, the developed method is the only one reported for MIF assembled on MMPs and addresses its determination in a relevant oncological scenario (paired non-tumoral (NT) and tumoral (T) tissues from individuals diagnosed with CRC at different stages of the disease). The analysis, requiring only 100 ng of tissue extract, allowed efficient discrimination between NT and T paired tissues, and successfully differentiated between healthy, early (I–II) and advanced (III–IV) CRC stages, achieving these results in just 105 min.

14667 An Intelligent Transvaginal Contained Bulk Tissue Extraction System – a Usability Study with 30 Surgeons

ABSTRACT Root‐knot nematodes ( Meloidogyne ), with their broad host range and ability to challenge plant resistance, represent a significant obstacle to the development of crop production, including eggplant cultivation. In this greenhouse study, the resistance of 26 eggplant varieties to M. javanica was evaluated. The plant seeds were sown in two‐kilogram plastic pots and kept under greenhouse conditions at a temperature of 27°C ± 2°C. The seedlings were inoculated at the four‐leaf stage with 6000 nematode eggs. After 60 days, the plants were harvested, and the indices of plant growth and nematode populations were evaluated. In addition, catalase, peroxidase, and superoxide dismutase activity were measured. For this purpose, the varieties Noorabad and Black Beauty, which are highly sensitive and less sensitive varieties, respectively, were planted in one‐kilogram pots and kept in the greenhouse. The seedlings were inoculated with 3000 nematode eggs at the four‐leaf stage. Root tissue extraction was performed at six time points (0, 5, 9, 13, 17 and 21 days) after inoculation. The results showed that the growth indices of the varieties Faselis and Black Beauty were better compared to the other varieties. The significant changes in enzyme activity observed in the Black Beauty variety in response to infection were considered to be the reason for the lower nematode damage. However, according to the Canto‐Saenz classification, all varieties analyzed were classified as susceptible, as they had a gall index &gt; 2 and a reproduction factor &gt; 1.

Exidavnemab binds to aggregated α-synuclein in human brains affected by α-synucleinopathies.

14609 An Intelligent Transvaginal Contained Bulk Tissue Extraction System – a Microbiological Test to Evaluate the Integrity of the Claria System