Heteromeric β2-containing nicotinic acetylcholine receptors (nAChRs) regulate diverse neural processes, yet their study in native circuits has been limited by the inability to manipulate receptor function with cell-type specificity, without transgenics, and across species. Existing tethered-ligand approaches, including photoswitchable and knock-in-based systems, provide valuable control of engineered receptors but do not readily integrate with Cre-driver lines and cannot be used in rats, where many nicotine-related behaviors are more robust. To address these limitations, we developed Cre-dependent adeno-associated viral (AAV) vectors expressing the β2 E61C nAChR subunit, a cysteine-bearing variant that enables covalent antagonism by the maleimide-PEG4-choline tethered ligand MPEG4Ch. We generated AAVs encoding either β2 E61C or hypersensitive β2Leu9'Ser E61C subunits and validated their functional incorporation into native nAChR pentamers in mouse and rat ventral tegmental area (VTA) neurons. Patch-clamp recordings demonstrated that MPEG4Ch produced strong, long-lasting, and E61C-dependent suppression of ACh-evoked currents, with near-complete blockade following prolonged ligand exposure. The system performed robustly in both DAT-Cre mice and rats receiving co-infused AAV-Cre, establishing effective cross-species use. From our results, we infer that any heteromeric receptor containing ≥1 engineered β2 subunit becomes ligand-sensitive, and that viral expression efficiently converts the endogenous β2 receptor pool without increasing total receptor abundance. This AAV-based, Cre-dependent E61C system provides a practical, optics-free, and species-flexible platform for cell-type-specific silencing of β2-containing nAChRs. Its compatibility with widely used Cre lines and suitability for rat behavioral paradigms-including nicotine self-administration and sustained-attention tasks-positions it as a versatile tool for dissecting nicotinic signaling in intact neural circuits.
Autosomal Dominant Polycystic Kidney Disease is caused by loss-of-function mutations in PKD1 or PKD2 genes, leading to reduced polycystin protein levels. Increasing PKD1 expression via CRISPR activation (CRISPRa) represents a promising therapeutic strategy; however, delivery of large CRISPRa plasmids into renal epithelial cells, and particularly primary cells, remains inefficient due to size-related barriers. We aimed to enable Pkd1 transactivation by miniaturizing CRISPRa plasmids into ~ 6 kb vectors using a one-pot method to enhance cellular uptake in mouse kidney epithelial cells. Using type IIS restriction enzymes, we excised the mammalian expression cassette from full-length large 9-11 kB plasmids. The excised cassette was engineered to have complimentary overhangs. Thermocycling with T4 DNA ligase promoted circularization of the excised cassette (forming ~ 6kB mini-CRISPRa vectors), and T5 exonuclease digestion removed residual backbone fragments. These mini vectors substantially enhanced nucleofection efficiency from 16.10% ± 0.53 to 54.17% ± 2.10 in Pkd1RC/- cells, and from 10.14% ± 1.40 to 31.27% ± 0.12 in primary Pkd1RC/Cond; Pkhd1Cre+ cells. Functionally, the mini-CRISPRa plasmid (mdCas9-VPR) with Pkd1-targeting sgRNAs induced robust endogenous Pkd1 upregulation compared with non-targeting controls: a 4.1-fold increase in Pkd1RC/- cells (p < 0.001) and a 2.9-fold increase in primary cells (p < 0.001). Full-length plasmids produced no significant activation in either cell type. Miniaturization of CRISPRa vectors with this one-pot approach overcomes delivery limitations in hard-to-transfect renal epithelial cells and enables efficient, functional Pkd1 activation, in vitro.
This study aimed to explore whether CD74 participates in regulating ferroptosis and to clarify the related mechanisms in traumatic brain injury (TBI). A TBI rat model was generated using controlled cortical impact. The ferroptosis inducer RSL-3, the inhibitor Liproxstatin-1 (Lip-1), and lentiviral vectors targeting CD74 or Nrf2 were injected into the lateral ventricle. Knockdown efficiency of the lentiviral vectors was verified by RT-qPCR. Motor performance was evaluated using the foot fault test, neurobehavioral function via mNSS scoring, brain water content using the wet-dry method, iron deposition in cortical tissues by Perls' Blue staining, Fe2+ levels with an iron assay kit, degenerating neurons by Fluoro-Jade C staining, and Nrf2/HO-1 pathway protein expression via Western blot. TBI rats displayed increased foot faults, elevated mNSS scores, increased brain water content, higher Fe2+ levels, more iron-positive cells, and greater numbers of degenerating neurons in the cerebral cortex. Lip-1 or CD74 downregulation alleviated TBI-related changes, whereas RSL-3 or CD74 upregulation worsened them. Downregulating CD74 enhanced Nrf2/HO-1 pathway activity, and Nrf2 knockdown counteracted the benefits of CD74 downregulation. Reducing CD74 expression ameliorates ferroptosis in TBI by activating the Nrf2/HO-1 signaling axis.
Phytoplasmas are cell wall-less bacteria that are transmitted by phloem-feeding insects. In Canada, insect vectors of this pathogen are leafhoppers (Hemiptera: Cicadellidae), and they can contribute to significant economic losses. As climate change alters the composition and movement of insect communities, migratory species such as the potato leafhopper (Empoasca fabae, Harris 1841), one of the most abundant leafhoppers in Québec affecting berries, may play an emerging role in phytoplasma transmission. Although E. fabae is not currently confirmed to act as a vector, its frequent presence and abundance in fields, along with its potential to acquire phytoplasmas, deserve further investigation. In this study, we tested DNA from E. fabae collected in strawberry fields for the presence of 'Candidatus Phytoplasma' using a highly validated nested PCR assay. The amplicon from positive insects were cloned and 46 of those clones were sequenced to identify phytoplasma groups and subgroups. Our findings confirmed the presence of multiple Aster Yellows (16SrI-related) subgroups in E. fabae, based on phylogenetic analysis, restriction fragment length polymorphism (RFLP) profiling, and single-nucleotide polymorphism (SNP) profiles. However, although phytoplasma was detected in a new generation of leafhoppers reared under controlled conditions in disease-free alfalfa plants, the ability of E. fabae to transmit the pathogen remains unknown. Overall, these findings highlight the importance of monitoring common pests such as E. fabae as early indicators of phytoplasma diversity in Eastern Canadian agricultural systems.
Cutaneous gene therapy has the potential to treat a wide range of skin disorders, but effective delivery remains limited by the skin's barrier properties and immune surveillance. Here, we identify AAVrh32.33 as a potent vector for targeting dermal stromal compartments. Following systemic administration in mice, AAVrh32.33 mediated robust and durable transgene expression, with preferential targeting of dermal fibroblasts and hair follicle bulge cells. Expression peaked at one month and persisted for up to two years, highlighting its suitability for chronic conditions. To reduce immunogenicity, a dominant CD8+ T cell epitope was disrupted, generating the IDPΔ variant. This modification attenuated peptide-specific T cell responses while preserving stromal transduction. In human skin explants, IDPΔ achieved high levels of gene expression, primarily in dermal fibroblasts and precursors, confirming translational relevance. Finally, vectors encoding CCL17, CCL20, and CCL22 demonstrated localized targeted therapeutic gene delivery in both healthy and inflamed skin, underscoring the feasibility of using this platform to reshape local immune responses. Together, these findings establish AAVrh32.33 and IDPΔ as promising platforms for durable cutaneous gene therapy, with direct applications in diseases such as vitiligo where long-term modulation of the dermal microenvironment is essential.
Tire related particles (TRPs) are widely distributed and critically important vectors for coexisting antibiotics in aquatic environments. As a major source entering surface waters, TRPs from wastewater treatment plants undergo disinfection and subsequent photoaging. Accordingly, this study investigates the combined effects of chlorination (a widely used disinfection method) and photoaging on TRPs' physicochemical properties and the adsorption of tetracycline (TC). Experimental observations suggest a possible synergistic ("1 +1 >2") effect where the combined chlorination and photoaging may produce a more pronounced aging effect than either process alone. Adsorption isotherm and adsorption kinetics suggested a tendency for moderate aging enhances adsorption, while excessive aging leads to a decrease in the overall adsorption affinity. Molecular dynamics simulations demonstrated interplay between decreased compactness, increased surface heterogeneity, and hindered site accessibility, explaining the observed transition from enhanced to inhibited TC adsorption with increasing aging. Furthermore, experimental adsorption studies revealed TC adsorption may facilitate the release of some TRPs' additives. Density functional theory calculations supported this observation, indicating that when the interaction energy between the additive and the TRPs is less than that between TC and the TRPs, the additive is preferentially replaced by TC. Collectively, these findings highlight the synergistic effect of chlorination and photoaging and underscore the potential for TRPs additives to be released into the surrounding environment upon interaction with coexisting organic contaminants.
The regeneration of functional dentin is a critical clinical goal for preserving tooth vitality after injury, with odontoblastic differentiation of human dental pulp stem cells (hDPSCs) being central to this reparative process. While the long noncoding RNA H19 is recognised as a key regulator of dentin repair, its downstream regulatory network is complex and incompletely mapped. Beyond the previously established H19/miR-140-5p/BMP2 axis, this study identifies a distinct and parallel pathway in which H19 promotes odontoblastic differentiation by downregulating miR-103a-3p, which in turn targets the PIK3R1/AKT and KLF4 signalling cascades. Our findings reveal that these two downstream networks operate independently, further elucidating the multifaceted role of H19 in dentin regeneration. In this study, we used lentiviral vectors to stably overexpress H19 in hDPSCs. Bioinformatic analysis and dual-luciferase reporter assays were employed to validate the interactions between H19 and miR-103a-3p, as well as between miR-103a-3p and its target mRNAs, including phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) and Kruppel-like factor 4 (KLF4). And qPCR and Western blot were used to investigate the expression pattern of H19 and the potential signalling axis of H19 and key odontogenic markers. Then, alkaline phosphatase and alizarin red S staining were used to evaluate odontogenic differentiation capacity. Finally, a heterotopic pulp regeneration model was established. And HE staining, Masson staining, immunofluorescence and immunohistochemistry were performed to verify the mechanism of H19 regulating odontogenic differentiation in vivo. In vitro, H19 promoted odontogenic differentiation of hDPSCs, while miR-103a-3p inhibited them. Both PIK3R1 and KLF4 were identified as direct targets of miR-103a-3p. Ectopic expression of either PIK3R1 or KLF4 restored the odontogenic differentiation capacity of hDPSCs suppressed by miR-103a-3p. Mechanistically, PIK3R1 promoted odontogenesis by activating the PI3K/AKT signalling pathway, whereas KLF4 acted independently as a transcriptional regulator. In vivo, H19 overexpression drove odontoblastic differentiation of hDPSCs by inducing the expression of its downstream targets, PIK3R1 and KLF4. Our findings indicated that H19 promoted odontogenic differentiation of hDPSCs by modulating the miR-103a-3p-PIK3R1/AKT and miR-103a-3p-KLF4 axes, underscoring their therapeutic potential for pulp regeneration.
Peripheral blood from four male patients with corticosteroid-related central serous chorioretinopathy (CSC) was used to generate human induced pluripotent stem cells (hiPSCs). Non-integrating episomal vectors were employed to deliver the reprogramming factors. All hiPSCs displayed typical morphology, expressed markers of the undifferentiated state, demonstrated the ability to differentiate into the three-germ layers, and exhibited normal karyotypes. These hiPSC lines offer a valuable model to study CSC pathogenesis.
Reptiles often inhabit environments that are in close proximity to humans and livestock, creating opportunities for parasite transmission. They are common in areas where they find shelter, food and warmth. The Bengal monitor lizard (Varanus bengalensis), a member of the family Varanidae, represents one of the largest groups of extant poikilothermic predators. Monitor lizards are known to harbor several tick species that serve as vectors for a variety of pathogens. No prior information is available in the literature regarding ticks infesting V. bengalensis in Pakistan as well as regarding the occurrence of Toxoplasma gondii in these ticks. Therefore, we aimed to determine the molecular prevalence of T. gondii in Amblyomma gervaisi ticks (n = 93) collected from 24 V. bengalensis in Buner District, Khyber Pakhtunkhwa Province, Pakistan, between May and September 2023. Polymerase chain reaction (PCR) amplified a 300 bp fragment specific for the ITS-1 region of T. gondii in 10 of the 93 (11%) A. gervaisi ticks. DNA sequencing and BLAST analysis confirmed the presence of T. gondii. Phylogenetic analysis showed that these sequences clustered with the ITS-1 sequences of T. gondii detected in reptiles and mammals from Pakistan, Brazil, China, Tunisia and Portugal. The prevalence of T. gondii in A. gervaisi was not limited to a specific tick sex, feeding stage or month of sampling. However, among the tick developmental stages, nymphs had the highest rate of T. gondii infection. In conclusion, for the very first time from Pakistan, we are reporting the presence of T. gondii in A. gervaisi that were infesting monitor lizards. We recommend that similar and large scale studies should be conducted in all those areas of Pakistan that are unexplored for the presence of T. gondii in A. gervaisi ticks. Prevalence of this parasite should also be screened in all the animals harboring these as well as other tick species. This will help in better understanding of T. gondii transmission to new hosts that will lead toward its effective control.
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder distinguished by progressive motor neuron degeneration, with diverse clinical manifestations and complex genetic and environmental triggers. The variability in disease progression underscores the necessity for tailored diagnostic and therapeutic approaches. MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression, have emerged as promising biomarkers and therapeutic targets in ALS. Dysregulation of specific miRNAs has been linked to mechanisms of ALS, including neuromuscular dysfunction, neuroinflammation, and neuronal survival/apoptosis. The potential of miRNA-based therapies, such as mimics and inhibitors, offers a more integrated approach by modulating entire disease networks, rather than targeting isolated pathways. However, challenges persist, particularly in delivering these therapies efficiently across the blood-brain barrier and minimizing off-target effects. Current delivery strategies involving nanoparticles, viral vectors, and exosome-based approaches require optimization for clinical use. This review synthesizes the latest research on miRNA-mediated mechanisms in ALS, evaluating their diagnostic, prognostic, and therapeutic potential, while highlighting the current limitations in clinical validation. It underscores the importance of standardized methodologies, multi-omics integration, and rigorous validation to facilitate the clinical translation of miRNA-based strategies. Standardized protocols and multicenter validation in large cohorts are essential to confirm the diagnostic accuracy of miRNAs, paving the way for their clinical application in ALS precision medicine.
Mosquitoes are vectors of deadly, life-threatening diseases worldwide. There is limited information on the interactions between microbes and local mosquito fauna in the Nigerian ecotype and Osun State. This study employed molecular techniques to characterize microorganisms isolated from the internal tissues of adult mosquitoes reared from larvae and pupae collected from various breeding sites in the Osogbo metropolis, Nigeria. Bacteria and yeasts were isolated from immobilized, surface-sterilized, and homogenized mosquitoes. Molecular identification of microbes was based on the Polymerase Chain Reaction (PCR) technique and sequencing of the 16S rDNA gene and internal transcribed spacer (ITS) region for bacteria and yeasts, respectively. The genus Wolbachia was screened with PCR using Wolbachia-specific primers. The adult mosquitoes harboured bacteria, namely Enterobacter bugandensis, Staphylococcus haemolyticus, Staphylococcus capitis, Enterobacter hormaechei, Pantoea dispersa, Sphingobium yanoikuyae, and Aerococcus urinaeequi. Yeasts identified were Meyerozyma caribbica, Rhodotorula mucilaginosa, and Candida orthopsilosis. The genus Wolbachia was not detected in the mosquitoes. The bacteria and yeast isolates from this study can play important roles in the biology of mosquitoes. Notably, S. yanoikuyae possesses bio-degradative potential, as reported in previous studies. Hence, this underscores the need for further investigation of the role of Sphingobium species in mosquito resistance to insecticides.
Cis-regulatory elements (CREs) drive tissue- and cell-specific gene expression and are essential for safe, sustainable genetic control strategies in pest and vector insects, including the engineering of gene drives in the primary human-malaria vector Anopheles gambiae. Yet CREs remain poorly defined in mosquitoes due to limited computational tools and practical methods for identification and validation. We present a systematic in silico approach for CRE discovery, correlating targeted DNA-motif searches with gene expression, followed by frequency and distribution analysis within putative promoter regions. Applied to the A. gambiae germline, this approach identified hundreds of putative CREs significantly correlated with germline expression in one or both sexes, often linked to distinct sperm developmental stages and chromosomal locations, suggesting roles in broader regulatory mechanisms such as dosage compensation and meiotic silencing. When mapped onto pre-characterised germline promoters, CRE distribution aligned with regions associated with experimental expression patterns. Finally, we validated a top-ranked testis-enriched CRE using an in vivo dual-reporter assay, showing that mutation of conserved nucleotides drastically altered male germline expression. To the best of our knowledge this work provides the first nucleotide-resolution regulatory genome annotation of the A. gambiae germline, offering a transferable framework to aid promoter design for genetic control strategies against malaria mosquitoes and other insect pests.
This study aimed to develop a method for extracting acoustic features to assess left anterior descending artery (LAD) stenosis severity. Heart sound data were collected from 75 participants (10 diastoles per participant) using a high-signal-to-noise ratio micro-electro-mechanical systems stethoscope. The diastolic signals were preprocessed, and empirical wavelet transform was applied to decompose their power spectra into three modes (0-150, 150-500, and > 500 Hz). The spectral energies (e(1), e(2), e(3)) of these modes were analyzed, and support vector machine (SVM) and extreme gradient boosting (XGBoost) machine learning algorithms were used to classify LAD stenosis into mild (< 50%), moderate (50%-75%), and severe (> 75%). Spectral energies e(2) and e(3) significantly increased with stenosis severity, and XGBoost outperformed SVM, achieving a test accuracy of 0.8133 and areas under the curve of 0.9358, 0.9644, and 0.9580 for mild, moderate, and severe stenosis, respectively. Empirical wavelet transform-extracted spectral energies of e(2) and e(3), combined with XGBoost, effectively determine LAD stenosis degree, offering a non-invasive screening tool.
With the widespread cultivation of genetically modified (GM) maize, accurate detection of GM components has become critical. This study developed three end-to-end DeepSpectra models (V1-V3) to classify GA21 maize at six GM concentrations (blank, levels 1-5). Spectral data were acquired using a terahertz time-domain spectroscopy system. Outliers were removed using an isolation forest, and the spectral data were preprocessed with Savitzky-Golay smoothing, standard normal variate transformation, baseline correction, first derivative (FD), and second derivative. Three comparison models were constructed, a support vector machine (SPA-GS-SVM) and a random forest (SPA-GS-RF) based on successive projection algorithms (SPA) and grid search (GS), as well as a one-dimensional convolutional neural network. Experimental results showed that the DeepSpectraV2 model with FD preprocessing achieved the best classification accuracy of 96.56%, outperforming the best comparative models by 1.52 to 15.52% across other preprocessing methods. This study presents a novel approach for the rapid non-destructive detection of GM crops.
Soluble solids content (SSC) is a critical indicator for pear maturity evaluation and quality grading. This study aims to develop a rapid, nondestructive, accurate and interpretable SSC prediction framework by coupling hyperspectral imaging (HSI), broad learning system (BLS), and Shapley additive explanations (SHAP). Raw spectral data of 'Qiuyue' pears were collected and preprocessed. A BLS prediction model was constructed based on the preprocessed full-spectrum data, with its performance compared to partial least squares regression (PLSR), backpropagation neural network (BPNN), and support vector regression (SVR). SHAP was adopted for BLS model interpretability analysis and key wavelength selection. Simplified prediction models were further established by applying BLS, PLSR, BPNN, and SVR to the key wavelengths selected by SHAP and three traditional wavelength selection methods. The results showed that BLS outperformed the other three models in both full-wavelength and key-wavelength modeling, with the full-wavelength BLS model achieving the optimal performance (prediction correlation coefficient RP = 0.8195, root mean square error of prediction RMSEP = 0.8037). In addition, SHAP effectively screened key wavelengths and quantified the independent contribution of each to SSC prediction, greatly improving the transparency and interpretability of the BLS model. This study confirms the feasibility of the HSI-BLS-SHAP strategy for nondestructive and interpretable pear SSC evaluation, providing a technical reference for nondestructive intelligent fruit quality detection.
The production of monoclonal antibodies (mAbs) in Chinese hamster ovary (CHO) cells is often affected by position-effect variegation and the gradual loss of transgene expression over time. Hence, we have designed a dual-promoter IgG expression vector and compared versions that either contained or lacked a CHO-derived matrix-attachment region (MAR). Stable CHO-S pools, cultured in serum-free conditions, revealed that the MAR-containing construct produced higher and more consistent antibody levels across ten passages, as confirmed by Western blot and Protein A Octet analysis. Product-quality analysis by size-exclusion chromatography and reducing SDS-PAGE confirmed formation of properly assembled, mainly monomeric antibodies in both cases. Quantitative PCR indicated greater transgene copy numbers in MAR pools (+ 48% for the light chain and + 71% for the heavy chain), and RT-qPCR showed roughly fourfold higher transcript levels for both chains relative to controls. Bioinformatic analysis revealed several SATB1 binding motifs within the MAR sequence, and ChIP-qPCR demonstrated SATB1 association with the MAR-linked transgene locus. Overall, the data suggested that a CHO-native MAR could enhance transgene dosage and transcriptional activity, while preserving product integrity, possibly through SATB1-mediated chromatin organization. Ongoing work includes chromatin-mark profiling and process-level productivity measurements to better define the impact of MAR-based vector design on biomanufacturing performance.
Differentiating progressive supranuclear palsy (PSP) from Parkinson's disease (PD) can be clinically challenging. In the neuroimaging field, radiomics has emerged as a promising approach to capture subtle microstructural and textural image alterations, improving differential diagnoses. To assess the diagnostic value of brainstem radiomic features from T1-weighted magnetic resonance imaging (MRI) in distinguishing PSP from PD patients. This study included 433 participants from two independent cohorts: an Italian training cohort (84 PSP and 177 PD) and an international validation cohort (68 PSP and 104 PD). Radiomic features including first-order, shape, and texture descriptors were extracted with PyRadiomics from brainstem segmentations generated by the automated deep-learning-based AssemblyNet pipeline. Classification models (Decision Tree, Support Vector Machine, Random Forest, and XGBoost) were trained using nested cross-validation and tested on the independent cohort. Model interpretability was examined with SHapley Additive exPlanations. Radiomics-based models yielded high and consistent performance in distinguishing PSP from PD, higher than brainstem volume. In the validation cohort, Random Forest and XGBoost achieved the best performance (area under the curve [AUC]: 0.93 and 0.94, respectively). Texture- and intensity-based radiomic features emerged as the most informative predictors, while shape descriptors showed lower relevance in discrimination between PSP and PD. Brainstem radiomics extracted from routine T1-weighted MRI demonstrated excellent classification performance in distinguishing PSP from PD patients and generalized robustly across independent datasets. Texture-based features captured microstructural disorganization not reflected by automated volumetry, underscoring the added value of radiomics for differential diagnosis in atypical parkinsonism and for integration in future multimodal biomarker frameworks. © 2026 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
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Viruses often hijack host developmental programs to promote infection, but the mechanistic links between reproductive regulation and antiviral immunity remain incompletely understood. Here, we identify a virus-triggered hierarchical degradation cascade that links antiviral immunity and fertility regulation in rice. We show that the rice grassy stunt virus (RGSV) effector P3 transcriptionally activates P3IP1, a RING-type E3 ubiquitin ligase. P3IP1 ubiquitinates and destabilizes the receptor-like cytoplasmic kinase RLCK22, which functions as a scaffold to stabilize the floral MADS-box transcription factors MADS1 and MADS15. The loss of RLCK22 results in decreased MADS1/15 protein levels, accompanied by reduced pollen viability and increased susceptibility to viral infection. Genetic and biochemical analyses support the existence of a regulatory module involving P3IP1, RLCK22, and MADS1/15. Mutants of mads1, mads15, or rlck22 exhibit overlapping molecular and antiviral phenotypes, including altered pollen viability and impaired transcriptional responses to RGSV. Our findings uncover a virus-inducible E3-RLCK-MADS axis linking post-translational regulation of development and defense, providing new insight into how pathogens manipulate plant fitness through targeted protein degradation.
Dengue, chikungunya, and Zika are Aedes-borne diseases (ABDs) of global health significance. Epidemiological studies with sensitive case detection are critical for evaluating vector control strategies to prevent ABDs; however, data on which surveillance method is most effective are limited. The performance of five surveillance methods (home visits, phone calls, SMS reminders, toll-free phone line (TF) and the Ministry of Health surveillance platform) was assessed, and the clinical characteristics of ABD cases in the targeted indoor residual spraying trial, which quantified the efficacy of preventive indoor residual insecticide applications against ABDs in Merida, Mexico, were described. A cohort of 4,461 children was monitored over three transmission seasons (July-December 2021-2023), with surveillance methods rotated weekly to detect illnesses. Kaplan-Meier curves and log-rank tests compared the time from symptom onset to laboratory testing across methods. Analysis of variance, t- and χ2 tests assessed differences in utilization of surveillance methods across demographic factors. Of 1,902 illnesses detected, 920 (48.4%) met suspected ABD criteria; 825 provided blood samples (89%), and 422 (51%) were confirmed as ABDs. Dengue represented 70.4% of confirmed cases (n = 297/422). Among confirmed cases, clinical manifestations were diverse, with fever (>94%), myalgia (80-100%), and headache (70-100%) being most frequent. Twenty-seven patients (9%; n = 27/297) had dengue with warning signs. TF detected 55.2% (n = 233/422) of confirmed cases and achieved the fastest time to laboratory testing. These results demonstrate case detection can be optimized. TF proved effective in rapidly identifying symptomatic reports, underscoring the value of integrated, low-barrier reporting systems for early arboviral detection.