Search Results (187)

Bacterial disease caused by Aeromonas hydrophila is a major constraint in intensive sturgeon aquaculture. This study examined the intestinal, humoral, and hepatic responses associated with dietary or waterborne pretreatment with the host-derived probiotic Bacillus subtilis Ya3.1 during A. hydrophila infection in hybrid sturgeon (Acipenser schrenckii ♂ × Huso dauricus ♀). Absolute taxon-targeted qPCR, serum innate immune assays, intestinal 16S rRNA gene sequencing, and untargeted liver metabolomics were integrated to evaluate probiotic-associated protective responses. Ya3.1 pretreatment increased intestinal B. subtilis-associated gene copy numbers before challenge and reduced the post-challenge A. hydrophila-associated burden. Among surviving fish at 10 days post-infection, both probiotic-pretreated groups showed significantly higher alkaline phosphatase activity than the infected control group; the dietary pretreatment group also exhibited a significantly higher complement C3 concentration. Microbiota analysis revealed marked community restructuring in untreated challenged fish, characterized by enrichment of Aeromonas and other opportunistic taxa, whereas probiotic-pretreated fish showed reduced challenge-associated compositional displacement and relative enrichment of Bacillus-related taxa. Liver metabolomics further indicated that Ya3.1 pretreatment was associated with altered hepatic metabolic profiles, with glycerophospholipid metabolism emerging as a recurrent candidate pathway. Overall, Ya3.1-associated protection was associated with a lower pathogen burden, reduced microbiota displacement, partial preservation of humoral immune indices, and altered hepatic metabolic responses. Full article

Background: Carotid atherosclerosis is a recognised manifestation of systemic vascular disease, and its association with coronary artery disease (CAD) has been well described. However, previous studies have largely been conducted under conventional diagnostic conditions and have focused on carotid plaque, intima–media thickness, or simple present-versus-absent stenosis classifications, rather than duplex-derived haemodynamic markers across the spectrum of angiographic CAD burden. The COVID-19 pandemic and post-pandemic period changed referral patterns and created more variable cardiovascular presentations, including symptoms that could resemble or mask obstructive CAD. Therefore, we investigated whether the established association between carotid stenosis severity and CAD burden remains detectable in a diagnostically heterogeneous real-world cohort, and whether routinely available carotid duplex haemodynamic parameters provide a clinically relevant signal in this setting. Methods: This single-centre, cross-sectional study was performed as a carotid-focused secondary analysis of the BG Study cohort. We included 902 consecutive patients who underwent invasive coronary angiography between 2021 and 2023 and carotid duplex ultrasonography during the same hospitalisation. CAD burden was defined according to the number of major coronary vessels with ≥70% diameter stenosis and classified as no CAD, one-vessel, two-vessel, or three-vessel disease. Carotid duplex parameters included peak systolic velocities of the common, internal, and external carotid arteries, as well as ICA stenosis severity graded according to NASCET criteria. Associations with CAD burden were assessed using a staged statistical approach combining χ² tests, Kruskal–Wallis tests with post hoc pairwise comparisons, Spearman correlation, inverse probability weighting, and ordered logistic regression. Results: The prevalence of measured ICA stenosis of any grade and severe ICA stenosis increased with greater CAD burden (both p < 0.001). Median PSV values of the bilateral ICAs and ECAs differed significantly across CAD groups on global intergroup testing. Post hoc pairwise analyses showed that significant corrected differences were concentrated between patients without CAD and those with multivessel or three-vessel CAD, particularly for ICA stenosis measures and bilateral ECA PSV. Spearman analysis demonstrated weak but statistically significant correlations between carotid parameters and CAD burden (ρ = 0.085–0.134). After inverse probability weighting, covariate balance was achieved, with all post-IPW standardised mean differences being <0.01. In ordered logistic regression (OLR) analysis, patient-reported history of carotid stenosis (OR 2.25, 95% CI 1.38–3.67; p < 0.001), right external carotid artery PSV per 10 cm/s (OR 1.31, 95% CI 1.09–1.57; p = 0.004), left ICA PSV per 10 cm/s (OR 1.17, 95% CI 1.01–1.36; p = 0.034), and left ICA stenosis per 10% (OR 1.24, 95% CI 1.11–1.39; p < 0.001) were independently associated with higher CAD burden. Exploratory ratio-based analyses showed that the ECA/CCA PSV ratio was associated with CAD presence and higher CAD burden, whereas the ICA/CCA ratio showed weaker associations; neither ratio-based index outperformed absolute ECA PSV. Conclusions: In this carotid-focused secondary analysis of a pandemic-era angiography cohort, carotid stenosis severity and duplex-derived haemodynamic parameters were independently but modestly associated with increasing angiographic CAD burden. These findings support carotid duplex markers as adjunctive indicators of systemic atherosclerotic burden rather than standalone tools for CAD detection or treatment decision-making. Future validation in vascular surgery populations is warranted to determine whether routinely available carotid duplex parameters can contribute to targeted cardiovascular risk recognition before major vascular procedures. Full article

Background/Objectives: One contemporary problem in health economics is the measurement and interpretation of socioeconomic inequalities in outcomes, utilisation, and resource distribution. This article aims to estimate socioeconomic inequality in the regional allocation of health resources in Bulgaria during 2019–2023. Methods: A year-by-year database was created. It includes regions (n = 28), population, GDP per capita, and the numbers of practicing physicians, dentists, nurses, midwives, hospital beds, and outpatient facilities. Income inequality is analysed using decile ratios, the Gini coefficient, the Generalised Entropy index, and the Atkinson index. Socioeconomic health inequality is quantified using the concentration index (CI) and the coefficient of variation (CV) of the absolute number and of resource density (per 1000 inhabitants). The socio-economic variable is a regional gross domestic product (GDP) per capita fractional rank and a frequency-weight approach to account for population size is used. The analysis is extended with the relative and slope indices of inequality. The CI of hospital beds, practicing physicians, and nurses is decomposed using the age dependency ratio and the number of hospitalisations by districts. Results: The Gini index levels remain stable, with no significant fluctuations, in the narrow range of 29.6–29.7. The highest inequality of the absolute resource’s quantity is among midwives (Mean CI = 0.498, CV = 0.018), and the lowest among nurses (Mean CI = 0.442, CV = 0.024). For material resources, a greater concentration of outpatient organisations in richer areas is observed (Mean CI = 0.481, CV = 0.035) than for hospital beds (Mean CI = 0.427, CV = 0.034). The dynamics and descriptives of inequality of resources’ density follow the same pattern, but with lower average rates, ranging from 0.045 to 0.112. The obtained estimates are statistically significant (p < 0.05). The analysis of the regression-based measures confirms, without any doubt, both the magnitude and the direction of the development of inequalities in the territorial distribution of health resources. Conclusions: Inequality measures vary by resource group. Significant inequality exists in the distribution of health resources between poorer and richer regions, particularly in material resources, in the outpatient sector. For most resource groups, a very slight decrease in inequality is observed midway through the analysed period. The most significant part of this inequality can be explained by differences in hospital care and income across richer and poorer regions. Full article

Decoration waste, because of its complex composition and the presence of volatile toxic and hazardous substances, has always been a difficult point in the management of urban construction waste. And with the continuous expansion of the town scale, the volume of decoration waste is gradually expanding, which constitutes a major challenge to the sustainable development of the construction industry. In order to solve this difficult problem, this paper took Henan Province as an example, and realized the accurate control of decoration waste based on GF-2 remote sensing images and a BP neural network model. The results of GF-2 remote sensing image interpretation and analysis showed that the spatial distribution of construction waste in the study area was extracted through a combination of manual visual interpretation and machine learning recognition, and as of 2021, the construction waste pile occupied a large proportion of the land area, of which the proportion of decoration waste was about 10%. Based on the trained BP neural network, the goodness-of-fit result was R = 0.95463. Selecting the research data from 2010 to 2021, the error of the predicted annual generation of decoration waste in Henan Province compared with the actual value was less than 15%, which had a high prediction accuracy. Based on the arithmetic sum of the projected figures for each year from 2022 to 2030, it is estimated that by 2030, the cumulative volume of construction and renovation waste generated in Henan Province will reach 49,827,200 tons. Visualization of spatial and temporal distribution characteristics was realized through ArcGIS, and the high production area of decoration waste was distributed from the beginning to the end of the distribution of multi-points to show the characteristics of a concentrated large area distribution, centrally located in southwestern and southeastern Henan Province, with the key cities of Zhumadian City, Luoyang City, Zhoukou City, and Xinyang City, which had obvious regional characteristics. At the same time, as the provincial capital, Zhengzhou has long ranked first in the province in terms of absolute case numbers and is therefore also a key focus of control measures. Uncertainty analysis indicates that the 95% confidence interval for the long-term forecast values is approximately ±12%. It is recommended to use the upper limit of this interval for the redundancy design of the absorption facilities to enhance the robustness of the decision. This study provides a theoretical basis and technical support for the governmental supervision of decoration waste during the development of national urban agglomerations, effectively solves regional urban planning and construction management problems, and promotes the sustainable development of the construction industry. Full article

In a steel continuous casting mold, argon bubbles injected through the submerged entry nozzle undergo transport, coalescence, and turbulent breakup, producing a polydisperse bubble swarm that affects flow stability and defect formation. In this study, an Euler–Lagrange model coupled with bubble collision coalescence and turbulence-induced breakup sub-models was established and validated using water model observations. Three daughter-bubble volume distribution models were compared in terms of bubble-cloud morphology, number-fraction distribution, and median-diameter evolution at different gas flow rates. For the median bubble diameter at different gas flow rates, the M-type model gives the lowest mean absolute error of 0.0349 mm. Large bubbles with diameters greater than 2.5 mm accounted for about 4% of the total number and were mainly concentrated near the SEN, whereas small bubbles with diameters of 1.0–1.5 mm accounted for about 60% and were dispersed throughout the upper recirculation region. Mechanism analysis further shows that bubble transport is drag-dominated in the high-velocity jet region, while buoyancy becomes more important in weaker flow regions; turbulent breakup is localized mainly in high-dissipation regions. Full article

Regulatory T cells (Tregs) play a key role in immune tolerance and are promising targets for treating immune-mediated diseases. This study investigated the direct effects of PEGylated graphene oxide nanoparticles (LP-GO, BP-GO at 5–25 μg/mL) and fullerenol C₆₀(OH)₂₄ (25–200 μg/mL) on human Treg viability and differentiation in vitro. Tregs were induced from peripheral blood CD4⁺ T cells using IL-2, TGF-β, and CD2/CD3/CD28 activation beads for 72 h with nanoparticles. Assessments included viability, apoptosis (Zombie aqua/Annexin V), phenotype (CD45⁺CD4⁺CD25⁺CD127^dim/−FOXP3⁺), nanoparticle sorption (intrinsic fluorescence), and IL-10 production. Neither PEGylated graphene oxide nor fullerenol C₆₀(OH)₂₄ affected T-helper (CD4⁺) viability (95.35–96.15%) nor early/late apoptosis levels. Despite this, we found a decrease in the percentage of CD4⁺ cells in cultures exposed to 50–200 μg/mL of fullerenol C₆₀(OH)₂₄. The percentage and absolute number of Treg cells decreased with 100–200 μg/mL of fullerenol, while IL-10 levels declined following treatment with 200 μg/mL of the same nanoparticles. Graphene oxide nanoparticles showed virtually no localization within or on cells. However, T helper and Treg cells demonstrated concentration-dependent sorption of fullerenol C₆₀(OH)₂₄ at concentrations of 100–200 μg/mL without a reduction in viability. These findings demonstrate good in vitro biocompatibility of the nanoparticles at pharmacological concentrations up to 25 μg/mL, alongside the inhibition of Treg differentiation with 100–200 μg/mL of fullerenol C₆₀(OH)₂₄. Full article

Background: Standardized operating procedures (SOPs) for intravenous vancomycin therapy have been shown to improve population-level trough target attainment and to reduce nephrotoxicity in orthopedic inpatients. However, mean target attainment on a population level does not capture how stably an individual patient remains within the therapeutic window. Intra-individual variability of vancomycin trough concentrations has remained underreported as a patient-level quality indicator in the orthopedic stewardship literature, despite its direct clinical relevance, as alternating sub- and supratherapeutic phases compromise both efficacy and safety independently of the mean exposure. Methods: We conducted a secondary analysis of the prospectively and retrospectively collected data of the Halle Vancomycin SOP cohort. Pre-SOP (n = 58) and post-SOP (n = 23) patient cohorts were compared with respect to patient-level variability metrics, including the coefficient of variation (CV%), swing index, mean absolute successive difference (MSSD), range of trough values, zone-transition frequencies, and the proportion of “stable” patients defined as CV% below 20%. First-order Markov transition matrices were computed to characterize the directionality of trough movements between subtherapeutic, target, and supratherapeutic zones. The primary analysis was restricted to patients with at least three documented trough measurements. Results: The median CV% decreased from 43.5% (IQR 33.5–51.5) pre-SOP to 32.5% (IQR 21.9–38.6) post-SOP (Mann–Whitney U, p = 0.011). The swing index decreased from 1.09 to 0.75 (p = 0.002), and the median range of individual trough concentrations shrank from 19.1 mg/L to 13.2 mg/L (p = 0.029). The absolute number of zone transitions per patient did not differ significantly between cohorts, but their directionality differed substantially: target-zone persistence increased from 37.8% to 57.6%. Across all 403 measurements, subtherapeutic values declined from 38.5% to 26.6%, while target-zone measurements rose from 28.5% to 44.7%. In the post-SOP cohort, longer therapy duration was associated with lower CV% (Spearman ρ = −0.52, p = 0.032). Conclusions: In addition to improvements in population-level target attainment, implementation of the SOP was associated with stabilization of the individual exposure profile of orthopedic inpatients receiving intravenous vancomycin. Intra-individual variability was lower in the post-SOP cohort, and transitions between zones were more often oriented toward the target range. These findings, derived from a single-centre secondary analysis with a small post-SOP cohort, support patient-level variability metrics as a complementary quality indicator in protocolized vancomycin management and warrant prospective multicentre validation. Full article

Introduction: Orthobiologics such as Platelet-Rich Plasma (PRP) and Injectable Platelet-Rich Fibrin (i-PRF) have emerged as promising tools in regenerative medicine. However, the lack of methodological standardization and the still limited comparative characterization between these products represent significant barriers to their optimized clinical application. This comparative laboratory study aimed to characterize and differentiate PRP and i-PRF, focusing on their cellular composition, obtained volume, and total Platelet-Derived Growth Factor (PDGF-BB) content. Materials and Methods: This study was conducted with 34 individuals meeting standard blood donation criteria. Peripheral blood samples were collected from all participants. PRP was obtained using a modified double-spin centrifugation protocol, whereas i-PRF was prepared using a modified low-speed centrifugation technique. Cellularity (platelet and leukocyte counts), final produced volume, and total PDGF-BB content were assessed using complete blood count analysis and an enzyme-linked immunosorbent assay (ELISA), respectively. Statistical analysis was performed using Linear Mixed Models (LMMs). Results: Both protocols resulted in significant increases in platelet and leukocyte concentrations compared to baseline values. PRP showed significantly higher platelet and leukocyte concentrations compared with i-PRF, as well as markedly higher PDGF-BB levels. In contrast, i-PRF yielded a substantially greater final volume and enabled a higher absolute delivery of total leukocytes, whereas PRP delivered a greater absolute number of platelets. In exploratory analyses, female sex, the presence of comorbidities, and increased abdominal circumference were associated with variations in product volume and cellular composition. Discussion: These findings indicate that PRP and i-PRF exhibit distinct biological profiles in terms of cellularity, volume, and total PDGF-BB content. Whether these laboratory differences translate into distinct clinical outcomes remains unknown. The results should therefore be viewed as hypothesis-generating: they suggest that PRP and i-PRF may not be interchangeable, and that future randomized clinical trials are needed to define product-specific indications based on the target tissue and desired biological mechanism. Full article

Cancer is a leading cause of death in humans and dogs. Several erythrocyte and platelet characteristics (indices and morphology) have shown promise as indicators of metastasis in humans. Similar studies have not been performed in dogs. This study evaluated erythrocyte and platelet characteristics measured on the Advia 2120i in 59 tumor-bearing dogs with carcinoma or sarcoma. Tumor-bearing dogs with and without intracavitary hemorrhage that underwent complete post-mortem and histopathology examinations were compared to healthy age-controlled dogs. Carcinoma- and sarcoma-bearing dogs without hemorrhage were compared. All tumor-bearing dogs without hemorrhage or metastasis were compared to those with metastasis, and characteristics were evaluated as indicators of metastasis. Tumor-bearing dogs without intracavitary hemorrhage (n = 49) had decreased hematocrit (p = 0.002) and reticulocyte hemoglobin content (p = 0.022), and increase in anisocytosis (p = 0.002), polychromasia (p = 0.002), macrocytosis (p = 0.032), codocytes (p = 0.022), absolute reticulocyte count (p = 0.035), platelet concentration (p = 0.002), plateletcrit (p = 0.022), and platelet volume distribution width (p = 0.022) compared to healthy dogs (n = 20). In tumor-bearing dogs with intracavitary hemorrhage (n = 10), additional significant differences were reflective of acute hemorrhage. No difference in characteristics between carcinoma- and sarcoma-bearing dogs without hemorrhage was identified. After correction for multiple comparisons, no differences in erythrocyte or platelet characteristics were identified between tumor-bearing dogs without intracavitary hemorrhage and metastasis and those without metastasis. Significant differences in characteristics exist between tumor-bearing dogs and healthy dogs. Based on the limited number of dogs in this preliminary study, no red blood cell or platelet characteristics were associated with metastatic disease in tumor-bearing dogs without hemorrhage. Full article

The jalapeño pepper (Capsicum annuum L.) is a crop of great economic and nutritional importance worldwide; however, increasing yield and quality under conditions of reduced synthetic inputs remains a significant challenge, mainly due to restrictions in plant nutrition and stress response capacity; in this context, plant-based biostimulants, such as Ricinus communis extracts, are of particular interest due to their potential to modulate plant metabolism, promote growth, and favor the accumulation of bioactive compounds. In this study, the effect of a foliar-applied biostimulant derived from a methanolic extract of Ricinus communis L. on the physiological, agronomic, and biochemical parameters of jalapeño peppers was evaluated under open field conditions. A randomized complete design with five treatments was established: three extract concentrations (T50: 50 mg L⁻¹, T75: 75 mg L⁻¹, and T100: 100 mg L⁻¹), a commercial biostimulant (Pepton 85/16 ^®), and an absolute control. Significant differences (α ≤ 0.05) were observed between treatments T50, T75, and T100 with the application of castor bean and the absolute control in stem diameter, fruit number, yield, and polar and equatorial fruit diameter, as well as phenols, flavonoids, and antioxidant capacity (ABTS and DPPH). The application of R. communis extract (T50, T75, and T100) significantly improved plant performance compared to the control, particularly in yield (up to 270%), fruit number (73%), shoot biomass (up to 38%), and root development (up to 32%). Furthermore, increases in chlorophyll content and in antioxidant-related compounds were observed, including phenols, flavonoids, ABTS, and DPPH (up to 17%). Spearman correlation analysis revealed strong associations between structural and metabolic variables, highlighting the relationship between stem diameter, fruit traits, and bioactive compound accumulation, as well as the link between chlorophyll content and reproductive performance. The ¹H NMR analysis indicated the presence of secondary metabolites such as ricin, unsaturated fatty acids, and phenolic compounds; however, their isolation and relationship with the biostimulant activity of the extract require further specific studies. Overall, foliar application of R. communis extract improved the growth, productivity, and biochemical attributes of jalapeño pepper, highlighting its potential as a sustainable alternative for crop management. Full article

Surface water systems are increasingly exposed to multiple pressures generated by climate variability, intensified water resource exploitation, and evolving geopolitical dynamics. This study provides a novel contribution by identifying critical threshold effects and non-linear interactions that influence nitrate concentrations through an integrated information systems framework. It develops an integrated information-system-based analytical framework that combines hydrological, climatic, geopolitical, and strategic indicators to shape the broader contextual framework within which hydrological and climatic pressures operate, rather than serving as direct predictors. Considering the nitrate concentration in rivers as a key parameter of water quality, the paper goes beyond univariate analysis of nitrite concentration, examining its relationship with four explanatory variables: the Water Exploitation Index Plus (WEI+), the number of heat stress days (Heat_Stress), the Geopolitical Risk Index (GPR), and a proxy variable representing the presence of strategic infrastructure (Nuclear_State) using a Reduced Error Pruning Tree (REPTree) decision tree algorithm with 10-fold cross-validation. The results indicate that climatic stress emerges as the primary predictor, with a critical threshold of approximately 7.83 heat stress days, beyond which nitrate concentrations increase significantly. Under conditions of high climatic stress and intensive water exploitation (WEI+ ≥ 67.39), predicted nitrate levels exceed 20 mg/L and can reach extreme values of up to 58.82 mg/L. In contrast, low hydrological pressure (WEI+ < 0.39) combined with moderate climatic stress is associated with very low nitrate concentrations, around 2.75 mg/L. The model demonstrates strong predictive performance, with a correlation coefficient of 0.976, a Mean Absolute Error (MAE) of 0.593, a Root Mean Squared Error (RMSE) of 2.046, and a Receiver Operating Characteristic (ROC) area exceeding 0.94 for classification tasks. While geopolitical and strategic variables do not act as direct predictors, they contribute to shaping the contextual framework influencing water resource management and environmental vulnerability. Overall, the study highlights the non-linear and systemic nature of water quality dynamics and demonstrates the effectiveness of decision tree-based models within integrated information systems for supporting environmental monitoring and decision-making under conditions of climate stress and geopolitical uncertainty. Full article

This study evaluated the effects of dietary spirulina (Spirulina platensis) supplementation on the physiological status and bone quality of juvenile ring-necked pheasants. A total of 200 one-day-old chicks were randomly allocated to a control group fed a basal diet or to an experimental group fed a basal diet supplemented with 15 g of spirulina/kg feed and reared under identical conditions for 42 days. Growth performance, feed intake, the feed conversion ratio, selected carcass traits, meat and bone quality, and biochemical and hematological parameters were assessed. Pheasants receiving spirulina presented significantly greater body weights at four weeks of age. The experimental group also exhibited increased pH values in pectoral and thigh muscles and increased postmortem muscle temperature. Spirulina supplementation significantly improved tibia quality, as reflected by enhanced geometric, structural, and material bone properties. The birds fed spirulina presented increased plasma total cholesterol and low-density lipoprotein concentrations, whereas those in the control group exhibited increased malondialdehyde levels, indicating increased oxidative stress. In addition, spirulina increased the proportion of lymphocytes and reduced both the proportion and absolute number of monocytes. In conclusion, spirulina supplementation increased tibia bone strength, reduced oxidative stress, and had immunomodulatory effects on juvenile pheasants. Full article

Antibiotic resistance genes (ARGs) are increasingly recognized as emerging contaminants in wastewater treatment systems; however, their responses to dissolved oxygen (DO)-limited conditions caused by insufficient aeration, particularly in the presence of microplastics, remain poorly understood. In this study, three sequencing batch reactors (SBRs) were operated for 31 days under progressively oxygen-limited conditions with different concentrations of polyethylene terephthalate (PET) microplastics to investigate their combined effects on treatment performance, microbial communities, ARGs, mobile genetic elements (MGEs), and PET degradation-related genes using metagenomic analysis. Prolonged oxygen limitation maintained relatively stable organic matter removal but progressively deteriorated ammonium removal and sludge settleability, while PET addition significantly aggravated these effects. PET exposure markedly increased the absolute abundance of ARGs without substantially altering resistome composition or dominant resistance mechanisms, suggesting an amplification rather than restructuring of the resistome. Correlation analyses indicated that ARGs enrichment was primarily host-associated and driven by the proliferation of a limited number of microbial taxa. Several potential ARG hosts were also strongly associated with PET degradation-related genes, indicating shared microbial populations linking PET-associated functions and antibiotic resistance. In addition, strong positive correlations between ARGs and MGEs suggested an important role of gene mobility in resistome dynamics under oxygen-limited conditions. Overall, these results demonstrate that oxygen limitation combined with PET microplastics promotes host-associated ARG enrichment in wastewater systems, highlighting potential environmental and public health risks and emphasizing the importance of maintaining operational stability to mitigate antibiotic resistance dissemination. Full article

The quantitative polymerase chain reaction (PCR) standard curve is the central analytical tool for validating qPCR assays and can also be used to estimate target concentrations in test samples. This review explains how qPCR standard curves are constructed, validated, and analyzed for different purposes. We first examine an idealized standard curve generated using an exceptionally high number of replicates, far exceeding typical routine use. This approach clearly illustrates fundamental qPCR characteristics and provides an educational framework for defining and estimating PCR efficiency, limit of detection, and limit of quantification. Furthermore, we demonstrate that, in theory, variation in threshold crossing points across replicates can be used to estimate the number of target molecules in a sample. This method, which we term variance PCR, could complement digital PCR and potentially extend the dynamic range of absolute quantification. We also analyze a representative standard curve as typically processed in routine qPCR workflows. This includes validating its dynamic range, assessing the impact of outliers, estimating PCR efficiency and precision, and finally applying the curve to determine the concentration of test samples. Full article

Global Positioning System (GPS)-derived ionospheric electron concentration measurements provide a powerful observational framework for seismo-electromagnetic studies, enabling quantitative investigation of lithosphere–atmosphere–ionosphere coupling processes through statistically detectable perturbations in ionospheric electron concentration. We analyze GPS-derived Vertical Total Electron Content (VTEC) variations associated with the 14 August 2021 Haiti earthquake ($M_w$ 7.2) and the 11 January 2022 Cyprus earthquake ($M_w$ 6.6) using data from nearby International GNSS (Global Navigation Satellite System) Service (IGS) stations located within their respective earthquake preparation zones. VTEC time series spanning 45 days before and 7 days after each event are processed to remove the diurnal component, yielding residuals that isolate short-term ionospheric variability. Anomaly detection is performed using three statistical frameworks: a Gaussian mean, standard deviation model, a robust median/median absolute deviation (MAD) model, and a distribution-free quantile-based model. Daily “occurrence” and “energy” indices are constructed to quantify the frequency and cumulative strength of detected anomalies, respectively. While the indices exhibit similar temporal patterns across all methods, they indicate frequent anomaly detection, limiting statistical selectivity. To address this, both indices are normalized by their median values and filtered using a 95% quantile threshold, retaining only extreme deviations. This procedure substantially reduces background fluctuations and isolates a small number of statistically significant anomaly peaks. For both earthquakes, enhanced anomaly activity is identified in the weeks preceding the events, whereas post-event peaks coincide with periods of elevated meteorological and geomagnetic activity. The results demonstrate that normalization combined with robust statistical methods is essential for discriminating significant ionospheric TEC anomalies from background variability. Full article