Baveno VII was introduced to help identify patients with compensated advanced chronic liver disease (ACLD) who should undergo screening gastroscopy to detect varices needing treatment (VNT), with a positive predictive value exceeding 90%. We performed a study at our centre to evaluate the impact of implementing this strategy, which has the potential to lower both healthcare costs and the risks associated with invasive procedures. We carried out a retrospective, single-centre analysis between 2021 and 2024, reviewing screening gastroscopies for oesophageal varices. Data reviewed included gastroscopy findings, transient elastography (TE) results performed within the preceding six months, and platelet count. The underlying cause of liver disease was also recorded. The primary aim was to evaluate the real-world application of the Baveno VII recommendations and estimate their impact on screening endoscopy utilisation and associated healthcare costs. We reviewed a total of 592 gastroscopies performed for variceal screening. Fifty-four patients were eligible for analysis after exclusion criteria were applied. Twenty-one of 54 patients (38.8%) did not meet the criteria for variceal surveillance. Notably, five out of these 21 patients (23.8%) were found to have VNT. Among the 33 patients who did meet the screening criteria, the positive predictive value for detecting VNT was 48.5% in our cohort. Applying the Baveno VII criteria would have excluded 16 patients who ultimately did not have VNT, potentiating cost savings of 29,120 Australian dollars (AUD). Application of the Baveno VII criteria in our cohort could have reduced unnecessary screening gastroscopies, resulting in cost savings and reduced procedural risk to patients. Although a small number of patients outside the screening criteria were found to have varices needing treatment, these findings should be interpreted in the context of the study's retrospective design, single-centre setting, and limited sample size. Nevertheless, this study provides a real-world example of the potential for Baveno VII-guided screening to reduce healthcare utilisation, costs, and patient exposure to invasive procedures.
Critically ill patients requiring treatment in the intensive care unit (ICU) suffer from muscle weakness that persists for years. As compared with healthy subjects, skeletal muscle of patients biopsied five years post-ICU revealed an abnormal transcriptome partially associated with poor muscle strength. We now hypothesized that skeletal muscle of long-term ICU survivors is "epigenetically aged", as determined by a muscle-specific epigenetic clock, and that such accelerated epigenetic aging contributes to their long-term muscle weakness. Muscle DNA-methylation data from former ICU patients at 5-year follow-up (N = 118) and healthy controls (N = 160), aged 18-89 years, were analyzed by the MEATv2 epigenetic clock. First, epigenetic age (DNAmAge), epigenetic minus chronological age (AADiff) and epigenetic age acceleration (AAResid) were compared between 97 former patients and 97 controls, propensity score-matched for age and sex. Next, the impact of any muscle-specific epigenetic aging of ICU survivors was investigated, via multivariable models, as a potential contributor to the altered transcriptome and reduced muscle strength. Former ICU patients showed a significantly higher muscle DNAmAge, AADiff, and AAResid than matched controls. In adjusted models, higher muscle DNAmAge, AADiff, or AAResid did not substantially contribute to differentially expressed muscle RNAs in former patients as compared with controls and was not associated with the poor long-term muscle strength. In conclusion, five years after ICU discharge, former patients showed accelerated epigenetic aging in skeletal muscle. However, the muscle-specific epigenetic clock did not capture molecular changes that are associated with long-term muscle weakness, which highlights the need for other muscle-specific biological predictors of age-related physical impairment. Trail Registration: ClinicalTrials.gov: NCT00512122.
Background Early inflammatory and fibrotic responses play important roles in tissue remodeling during wound healing. We developed a novel high-density collagen xerogel thread (CXT) and evaluated its effects on early urethral tissue responses in a rat urethral injury model. Methods A rat urethral incision model was used to compare CXT with conventional absorbable Vicryl® sutures (Ethicon, Inc., Somerville, NJ, USA). Histological evaluation was performed using hematoxylin-eosin staining and immunohistochemical analyses for alpha-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), and leukocyte common antigen (LCA) on postoperative day 7. Results Compared with Vicryl sutures, CXT was associated with reduced urethral mucosal thickening, lower numbers of CTGF-positive cells and LCA-positive cells, and less prominent stromal α-SMA expression during early wound healing. Conclusions CXT was associated with altered inflammatory and fibrotic tissue responses during early urethral wound healing in rats.
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Astragalus membranaceus is a Traditional Chinese Medicine (TCM) often used to modulate intestinal flora. This study aims to analyze the effects of Astragalus membranaceus on intestinal flora and chemotherapy-related complications in patients undergoing chemotherapy for colon cancer through clinical research and to explore the possible mechanism. The study included 86 patients with colon cancer who were assigned to the Chemotherapy (Che) and Astragalus (Ast) groups. The Che group was treated with bevacizumab combined with oxaliplatin and capecitabine and the Ast group was treated with Astragalus granules on the basis of the Che group. The intestinal flora and the incidence of chemotherapy-related complications were compared between the two groups. Astragalus granules can reduce a variety of complications during chemotherapy in patients with colon cancer. At the same time, this study detected the feces of patients before and after chemotherapy and found that Astragalus can effectively increase the content of intestinal Bifidobacterium and Lactobacillus and reduce the content of Enterococcus and Escherichia coli in patients. In addition, by detecting the serum of patients in this study, it was found that Astragalus granules can increase the level of serum CD4+ T cells and reduce the level of CD8+ T cells in patients. At the same time, Astragalus granules can also relieve the inflammatory state of patients during chemotherapy. TCM formula based on astragalus can effectively regulate the intestinal flora of patients, reduce the complications of chemotherapy and relieve the inflammatory state of the body during the chemotherapy of patients with colon cancer. Astragalus membranaceus has significant advantages in effectively improving the imbalance of intestinal flora caused by chemotherapy, improving the immune function of patients and reducing the incidence of chemotherapy-related complications.
Acute microglia-mediated neuroinflammation and oxidative stress play important roles in the pathogenesis of gas explosion (GE)-induced traumatic brain injury (TBI). Curcumin has documented neuroinflammatory protective properties; however, its effects on gas explosion (GE)-induced TBI and the underlying mechanism remain unclear. In this study, we established a male rat model of acute TBI and an in vitro model of acute microglial impact injury using shockwave physiotherapy. The effects of curcumin on the extent of the brain injury, as well as the levels of inflammatory cytokines, oxidative stress indicators, microglia polarization, and toll-like receptor 4 (TLR4) protein expression in model rats and microglial cells were evaluated using histological staining, western blotting, qPCR, immunohistochemistry, immunofluorescence, and biochemical assays. The results showed that curcumin reduced the pathological changes induced by GE and significantly inhibited the expression of neuron-specific enolase (NSE) (P < 0.05), a marker of microglial activation. Curcumin treatment also promoted M2 polarization of microglia (P < 0.05) and reduced the protein expression levels of TLR4, myeloid differentiation factor 88 (MyD88), NF-κB (P < 0.05), and the pro-inflammatory factors tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and NLRP3 (P < 0.05). Moreover, curcumin treatment significantly reduced the levels of oxidative stress factors in rat tissues (P < 0.05). In summary, our findings indicated that curcumin alleviated acute neuroinflammation and oxidative stress induced by GE in vivo and in vitro by inhibiting TLR4-mediated M2 polarization of microglia, thus providing a new treatment target for the neuroinflammation associated with TBI caused by explosives.
Thirteen new 4-acyl-5-amino-1H-imidazole alkaloids (1-13) were identified from cultures produced by the marine-derived Streptomyces sp. OUCMDZ-944 using feature-based molecular networking (FBMN)-guided isolation. Notably, compounds 1 and 2 were identified as phenyl-containing derivatives, specifically 5-amino-4-phenylpropionylimidazole (1) and 5-amino-4-phenylbutyrylimidazole (2). Ten isolates (4-13) are hydroxylated, with compound 13 being identified as a novel dimeric analogue. Furthermore, five methylated derivatives, 1a, 4a, 4b, 17a, and 17b, were synthesized. In an in vitro depression model, compounds 1, 4, 4a, 4b, 8, 16, and 17b were found to significantly reduce IL-1β levels, while compounds 1, 1a, 4b, and 10 notably reversed the corticosterone-induced decrease in brain-derived neurotrophic factor (BDNF) expression, suggesting a potential antidepressant-like activity. In the zebrafish anxiety model, compounds 1, 1a, 4b, 16, and 17b significantly enhanced spontaneous locomotor activity, reduced thigmotaxis behavior, and increased time spent in the inner zone under various lighting conditions, demonstrating clear anxiolytic-like effects. These five compounds were identified as promising lead candidates with dual anxiolytic and antidepressant potential.
We report the synthesis and characterization of a series of isomeric indacenodithiophenes (iso-IDTs) bearing various ethynyl substituents at the apical carbons. Absorption spectroscopy, cyclic voltammetry, and density functional theory (DFT) calculations were performed to investigate the electronic properties of these compounds. Notably, analogues with reduced steric protection (i.e., trimethylsilyl, adamantyl) were also successfully isolated and investigated. Computed nucleus-independent chemical shifts and ring current plot analyses reveal that iso-IDTs display reduced antiaromatic character compared to indenofluorene (IF) and the previously reported syn- and anti-fused IDT isomers, suggesting that the π-bond arrangement within the indacenodithiophene π-system can be leveraged for tuning their optoelectronic properties.
Acute decompensated heart failure (ADHF) remains a leading cause of hospitalization worldwide. The prognostic value of spot urinary sodium concentration in determining clinical outcomes for patients with ADHF treated with diuretics remains uncertain. This systematic review with meta-analysis was registered in the International Prospective Register of Systematic Reviews (CRD42024626912). MEDLINE, Cochrane, and Embase were searched from inception to May 2026 for studies comparing high and low urine sodium, measured after the first void or within six hours of diuretic use, in adult patients admitted for ADHF. The primary outcomes of interest were mortality, length of hospitalization/stay (LOS), inotrope use, and worsening renal function. Odds ratios (ORs) with 95% confidence intervals (CI) were pooled with a random-effects model. Quality assessment and risk of bias were performed according to Cochrane recommendations. A total of eight studies comprising 1,359 patients were included in the meta-analysis, of which 923 (67.9%) were categorized in the high urinary sodium group and 464 (32.1%) in the low urinary sodium group. In patients with ADHF, high urine sodium was associated with lower mortality (OR, 0.28; 95% CI, 0.12-0.68), reduced inotrope use (OR, 0.40; 95% CI, 0.25-0.62), lower worsening renal function (OR, 0.53; 95% CI, 0.32-0.86), and shorter LOS (mean difference, -4.35; 95% CI, -7.88 to -0.81) compared with low urine sodium. In patients with ADHF treated with diuretics, high urine sodium is associated with improved outcomes, including reduced mortality, inotrope use, worsening renal function, and shorter LOS, compared with patients with low urine sodium.
RNA interference (RNAi) provides a powerful tool for functional gene identification and pest management, and nanocarriers have recently enabled noninvasive transdermal RNAi delivery. However, the mechanism of nanocarrier-mediated double-stranded RNA (dsRNA) translocation across the insect cuticle remains unknown. We used a star cationic polymer (SPc) as a model to visualize this process and elucidate its interactions with aphid cuticle components. SPc self-assembled with dsRNA to form stable nanocomplexes via electrostatic and hydrophobic interactions. This assembly altered the wax layer morphology and increased chitin interfibrillar spacing, creating a permissive interface for dsRNA translocation. SPc also increased the contact area, reduced the contact angle, and significantly improved penetration through artificial wax/cuticle. Furthermore, SPc interacted with major cuticle components through various intermolecular forces, causing wax dissolution and reduced interfacial resistance at the chitin layer. These findings demonstrate that nanocarrier-mediated wax dissolution and chitin-layer barrier reduction facilitate dsRNA translocation across the insect cuticle, providing a theoretical foundation for transdermal delivery systems.
The fabrication of maxillary prostheses in patients with flap-reconstructed maxillary defects continues to present both clinical and technical challenges. Conventional impression techniques can be hazardous and often require multiple adjustments. This report presents a simplified digital approach using 3D scan data of an existing, well-adjusted prosthesis. The previously relined and adjusted prosthesis was scanned with an intraoral scanner, and the 3D surface data were used to print an occlusal record base. After recording the jaw relation, the teeth were arranged, and a functional impression was made at the try-in stage. Finally, the definitive prosthesis was fabricated using heat-cured acrylic resin. The printed record base provided accurate adaptation and minimized the clinical risks associated with conventional impression making. Incorporating the functional impression during the try-in stage further improved the fit and reduced the need for postdelivery adjustments. This technique offers a practical and simplified method for maxillary prosthesis fabrication. Reusing the morphology of a clinically adapted prosthesis eliminates complex digital processing and reduces clinical burden, making it especially useful in settings with limited access to advanced technical support.
Post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis is one of the important procedure-related complications of ERCP. Periprocedural intravenous hydration with lactated Ringer's solution has been considered a simple and feasible measure for reducing this complication. The present trial was designed to assess whether aggressive hydration with lactated Ringer's solution was more effective than standard hydration in preventing post-ERCP pancreatitis (PEP). This randomized controlled trial was performed at two tertiary care hospitals. A total of 126 patients undergoing first-time ERCP were included and randomly distributed into two equal treatment groups. Patients in group A were managed with standard hydration, whereas patients in group B received aggressive hydration using lactated Ringer's solution. The main outcome measure was the development of PEP. Other recorded outcomes were post-ERCP hyperamylasemia, isolated hyperamylasemia, serial pain scores on the visual analog scale, serum amylase levels, duration of hospital stay, and fluid-related adverse effects. All 126 randomized patients completed the 24-hour assessment. PEP developed in 14 patients in the standard hydration arm and four patients in the aggressive hydration arm (22.2% versus 6.3%; p = 0.020; RR = 0.29, 95% CI = 0.10-0.82). Total post-ERCP hyperamylasemia was less frequent after aggressive hydration than standard hydration (14.3% versus 41.3%; p = 0.001). Isolated hyperamylasemia was recorded in 7.9% and 19.0% of patients, respectively (p = 0.116). Pain scores remained lower with aggressive hydration at 4, 12, and 24 hours (all p < 0.001). Hospital stay was also reduced (1.8 versus 3.0 days; p < 0.001). Aggressive hydration with lactated Ringer's solution significantly reduced PEP, post-procedure hyperamylasemia, pain scores, and hospital stay compared with standard hydration. The regimen was well tolerated in selected patients without fluid-overload risk.
Lymphoma can seriously affect the quality of life of patients. Both homoharringtonine (HHT) and tanshinone IIA (Tan IIA) have shown robust anti-proliferation and apoptosis-inducing effects in lymphoma cells. However, it is unclear whether they can be used in combination to treat acute myeloid leukemia and lymphoma. Cell Counting Kit-8 (CCK-8) was employed to measure cell viability, the 5-Ethynyl-20-deoxyuridine assay (EdU) was used to quantify cell proliferation, and the transwell migration assay was used to assess cell migration in U937 and Raji cells. Additionally, transmission electron microscopy and nanoparticle tracking analysis assays were used to observe and identify exosomal structure, respectively. Combined HHT and Tan IIA treatment synergistically inhibited the viability and proliferation of U937 and Raji cells, induced apoptosis, and reduced vascular endothelial growth factor (VEGF) levels in these cells. Additionally, VEGFA and VEGFC levels were significantly reduced in exosomes derived from Raji cells that had been treated with HHT and Tan IIA (ExoHHT + Tan IIA). Moreover, absorption of ExoHHT + Tan IIA significantly inhibited the viability, proliferation, and migration of human umbilical vein endothelial cells by downregulating the expression of serine/threonine kinase 1 (Akt), matrix metalloproteinase-2 (MMP2) and MMP9. ExoHHT + Tan IIA suppressed the tumor microenvironment. HHT and Tan IIA combined therapy synergistically suppressed lymphoma progression by downregulating the Akt/MMP signaling pathway, suggesting their use in treating lymphomas. Clinical trial number: Not applicable.
Directed self-assembly (DSA) of block copolymer (BCP) thin films is a promising strategy for fabricating nanoscale patterns. However, defects and limited controllability during processing remain significant barriers to practical implementation. In this study, we introduce solvent immersion annealing (SIA) as a facile and effective approach to improving alignment quality and suppressing defect formation in shear-aligned polystyrene-b-poly(2-vinylpyridine) (PS-b-P2VP) thin films. By immersing shear-aligned films in solvent mixtures, SIA provides enhanced control over polymer swelling and chain mobility, enabling rapid annihilation of defects and long-range ordering within minutes. Systematic analysis of immersion time, solvent composition, and swelling behavior reveals that the optimal SIA process is achieved by preserving a delicate balance between chain rearrangement and film stability during solvent immersion. Compared to solvent vapor annealing (SVA), SIA offers superior swelling controllability and reduced sensitivity to external factors such as ambient humidity. Furthermore, large-area SEM and GISAXS analyses confirm that SIA enables scalable fabrication of well-aligned nanopatterns across extended surfaces. Taken together, this work highlights SIA as a robust and efficient strategy for achieving BCP nanopatterns with substantially reduced defect density, therefore offering potential for future lithography applications.
Postoperative infections (POIs) significantly contribute to morbidity and mortality following partial hepatectomy for hepatocellular carcinoma (HCC). While the Systemic Immune-Inflammation Index (SII) and Prognostic Nutritional Index (PNI) are recognized biomarkers for immune-inflammatory and nutritional status, their combined predictive value for POIs in liver surgery requires further investigation. This study evaluates SII and PNI as preoperative predictors for infections in this patient population. A retrospective observational study was conducted on 300 patients undergoing partial hepatectomy between 2022 and 2024. Preoperative laboratory data were used to calculate SII and PNI, with POIs identified within 30 days based on CDC guidelines. Statistical analyses, including multivariate logistic regression, were performed to compare infected and non-infected cohorts and identify independent predictors of infection. Of the 300 patients, 96 (32%) developed POIs. The infected group exhibited significantly higher SII (1142 ± 618 vs 792 ± 450) and lower PNI (40.1 ± 5.8 vs 46.4 ± 5.9) than the non-infected group. Multivariate analysis confirmed high SII (OR 2.85) and low PNI (OR 3.26; 95% CI, 2.01-5.12) as independent predictors. Furthermore, infections were associated with prolonged hospitalization, increased ICU admissions, and higher 30-day mortality. Preoperative SII and PNI are effective, independent predictors of POIs in patients undergoing hepatectomy for liver cancer. Integrating these biomarkers into routine evaluation enhances risk stratification and guides perioperative optimization. Early identification through these indices allows for targeted interventions, such as nursing-led nutritional support and intensified surveillance, to reduce complications and improve surgical outcomes.
Terrestrial insects underpin key ecosystem services, including pollination, herbivory regulation, decomposition, nutrient cycling, and disease control. These functions depend on chemical communication that guides insects to food, mates, hosts, shelters, and oviposition sites while helping them avoid threats. Environmental microparticles, such as micro- and nanoplastics, tyre wear particles, soot, mineral dust, and agricultural residues, are now widespread across air, soil, vegetation, and indoor environments, exposing insects through contact, deposition, and ingestion. Growing evidence shows that these particles disrupt insect olfaction by adsorbing volatile compounds, blocking antennal sensilla, and interfering with receptor and neuronal processes. These disruptions impair foraging, mating, oviposition, and host seeking, leading to reduced individual performance and declines in population density. As a result, insect-mediated services such as pollination, biological control, seed dispersal, and nutrient cycling are weakened, with consequences for plant community composition, biodiversity, food web stability, and crop yields. Despite these advances, the mechanisms linking microparticles to olfactory disruption remain poorly resolved, and no synthesis has integrated evidence across the full pathway from signal emission to neural processing. This review brings together chemical, physiological, and ecological evidence to map how microparticles interfere with insect olfaction across this pathway, identify key knowledge gaps, and propose a multi-level research framework to guide future work from molecular mechanisms to ecological consequences.
The thyroid gland and liver share a complex bidirectional relationship that is fundamental to metabolic regulation and hormonal homeostasis. Thyroid hormones (THs) regulate hepatic lipid handling, glucose metabolism, mitochondrial function, and energy balance, while the liver governs TH transport, activation, metabolism, and clearance. Increasing evidence links thyroid dysfunction with metabolic dysfunction-associated steatotic liver disease (MASLD), fibrosis progression, and adverse metabolic outcomes. This narrative review provides an updated synthesis of the mechanistic, clinical, and therapeutic aspects of thyroid-liver interactions and their implications for clinical practice. A comprehensive review of mechanistic, clinical, translational, and therapeutic studies examining thyroid dysfunction, liver disease, thyroid hormone sensitivity, and emerging thyroid hormone-based therapies was conducted. Thyroid dysfunction contributes to MASLD, dyslipidemia, insulin resistance, and hepatic injury, whereas liver disease alters TH metabolism and complicates the interpretation of thyroid function tests, including the occurrence of nonthyroidal illness syndrome. Emerging evidence suggests that reduced intrahepatic TH signaling and altered tissue-level hormone sensitivity play central roles in steatosis and fibrosis progression. Clinically, recognition of these interactions may improve the interpretation of thyroid abnormalities in liver disease and support risk stratification in metabolic liver disorders. The development of liver-targeted thyroid hormone receptor-β agonists, including resmetirom, represents a major therapeutic advance with potential to reshape management strategies for metabolic dysfunction-associated steatohepatitis (MASH). However, important controversies remain regarding the diagnostic utility of thyroid hormone sensitivity indices, long-term safety of thyromimetics, and the role of thyroid hormone replacement in liver-directed therapy, highlighting the need for robust prospective studies.
Mechanical strain is a powerful degree of freedom for modulating the thermoelectric performance of quantum structures. Herein, combining density functional theory and the nonequilibrium Green's function method, we systematically investigate the strain-mediated thermoelectric regulation of blue phosphorene nanoribbon heterojunctions (BPNRHJs). The results demonstrate that strain effectively tailors the electrical conductance of monolayer and stacked bilayer BPNRHJs. Specifically, strain shifts the conductance peaks toward the Fermi level at negative chemical potentials and substantially amplifies peak conductance values at positive chemical potentials. Moreover, strain modulates the position and profile of anti-resonant transmission dips induced by destructive quantum coherence, thereby tuning the peak position and enhancing the magnitude of the Seebeck coefficient. Additionally, strained structures exhibit suppressed phonon transmission spectra and reduced phonon transmission coefficients, leading to decreased phonon thermal conductance. Benefiting from the optimized electronic and phononic transport properties, the thermoelectric figure of merit (ZT) is significantly improved. At 500 K, applying strain from 0 to 0.2 GPa increases the maximum ZT from 1.4 to 2.5 for monolayer BPNRHJs and from 1.2 to 2.0 for bilayer BPNRHJs.
Older adults face multiple barriers that limit their engagement with social media. This systematic review aims to identify, categorize, and synthesize the barriers to social media adoption among older adults. A systematic search was conducted across six electronic databases without time restrictions. Studies were included if they involved participants aged 60+ years (or mean age 60+ years) and reported barriers to social media. Quality appraisal was performed using the Mixed Methods Appraisal Tool (MMAT). A total of 32 studies were included and synthesized thematically. Technical competency issues, including difficulties with device operation, updates, and Information and Communication Technology (ICT) literacy, were prevalent. Privacy and security concerns were consistently reported as critical obstacles. Educational support limitations, reduced learning speed, psychosocial factors (e.g., low self-efficacy), and socioeconomic constraints were also reported. This review synthesizes reported barriers and can inform the design of future studies and the development of strategies to promote digital inclusion among older adults.
Accurate quantification of aluminum (Al) uptake is essential for evaluating metal toxicity in experimental models. This study aimed to optimize pretreatment strategies and determine the effects of individual number and dose on Al accumulation and assess the biological relevance of Al exposure in the Caenorhabditis elegans model. The investigation was conducted in two stages. In the first stage, synchronized groups of 10, 25, 50, 100, 200, and 400 worms, along with controls, were exposed to 10, 15, and 25 mM AlCl₃ for seven days After exposure, worms were washed to remove Al from the culture medium, viability was terminated using nitric acid, and Al levels were quantified by ICP-MS without pretreatment. In the second stage, 50 worms exposed to 100 mM AlCl₃ for seven days and subjected to four different pretreatment protocols prior to ICP-MS analysis. Reproductive toxicity was additionally evaluated to determine the biological impact of Al accumulation. Significant dose-dependent increases in Al uptake were observed, particularly in groups containing 10, 25, and 50 worms (first trial). Among pretreatment methods (second trials), the combined sonication and centrifugation supernatant protocol yielded the highest Al levels (78.47 mM), followed by pretreatment A (68.68 mM), C (64.52 mM), and B (59.60 mM). Furthermore, Al exposure significantly reduced reproductive capacity compared with controls These findings demonstrate that both organism number and pretreatment strategy critically influence Al quantification and confirm that accumulated Al is associated with biologically relevant toxic effects. The optimized methodology provides a robust framework for future metal toxicity studies.