Highly pathogenic avian influenza H5N1 clade 2.3.4.4b viruses present a broad host range, with recent spillover and sustained transmission in dairy cattle reported in the USA. Replication-competent reporter viruses are critical tools that enable real-time monitoring of virus replication, facilitating high-throughput screens. In this study, we engineered three recombinant H5N1 clade 2.3.4.4b reporter viruses expressing nanoluciferase (NLuc) and two fluorescent reporter proteins, miniGFP2 and UnaG within the open reading frame of the nonstructural gene of the bovine A/Cattle/Texas/063224-24-1/2024 (TX2/24) virus. All reporter viruses replicated efficiently in vitro, presenting replication kinetics comparable to the parental rTX2/24 virus, but exhibited smaller plaque sizes, suggesting reduced cell-to-cell spread. In vivo infection studies in mice showed comparable pathogenicity among all four viruses, although rTX2/24-miniGFP2 and rTX2/24-UnaG exhibited decreased virus shedding relative to rTX2/24 and rTX2/24-NLuc. Virus titrations and in situ localization of virus replication sites demonstrated robust replication in respiratory tissues, with slightly attenuated systemic dissemination of all three reporter viruses. Fluorescent virus neutralization assays using miniGFP2 and UnaG reporter viruses accurately quantified neutralizing antibody titres in sera from naturally infected dairy cattle, consistent with wild-type virus assays. Additionally, the utility of the NLuc reporter virus for antiviral screening was validated against oseltamivir in vitro. Collectively, these results establish the H5N1 TX2/24-based reporter viruses as versatile and biologically relevant tools for investigating H5N1 pathogenesis and for use in serological and antiviral drug screens.
Thyrotropin-releasing hormone receptor 1 (TRHR1, encoded by Trhr, also referred to as Trhr1 in rodents) is a key component of the hypothalamic-pituitary-thyroid axis and has also been implicated in emotion, cognition, and stress-related regulation. However, the brain-wide distribution and cellular characteristics of Trhr1 reporter-labeled cells in the central nervous system remain incompletely understood. In this study, we generated Trhr-P2A-iCre; R26-tdTomato mice to genetically label Trhr1 reporter-labeled cells and combined this strategy with tissue clearing and volumetric imaging with synchronized on-the-fly scan and readout (VISoR) to construct a high-resolution brain-wide map. Quantitative analysis revealed that Trhr1 reporter-labeled cells were broadly distributed throughout the adult mouse brain, with marked regional heterogeneity. Higher densities were observed in the olfactory bulb, prefrontal cortex, striatum, hypothalamus, and amygdala, whereas lower densities were found in the hippocampus, thalamus, midbrain/brainstem, and cerebellum. Further immunofluorescence analysis indicated that Trhr1 reporter-labeled cells were predominantly neuronal in the examined regions, showing frequent co-localization with NeuN but not with Iba1 or GFAP. Many tdTomato-positive cells in selected cortical, amygdalar, and striatal regions co-localized with CaMKII, whereas overlap with PV or SST was limited; in the dentate gyrus, many co-localized with Prox1, indicating a granule-cell identity. Together, these findings provide a brain-wide anatomical map and region-specific cellular profile of Trhr1 reporter-labeled cells and establish a structural foundation for future studies of TRHR1-related neural circuits and functions.
In vivo monitoring of circadian rhythms depends on reliable and non-invasive detection methods. This is often achieved by expressing reporter genes heterologously under the control of a circadian promoter. The activity or fluorescence of the gene product is then used as a readout. To avoid the generation of such reporter strains, we recently established a reporter-free detection method for cyanobacterial batch cultures. To determine whether these rhythms are driven at the level of individual cells or result from population-based effects, such as gating of cell division, we analysed individual Synechocystis sp. PCC 6803 cells by combining a microfluidic cultivation technique with time-lapse microscopy imaging. Hundreds of time-lapse image sequences were processed using our deep learning cell segmentation workflow. Although the cells had been entrained by a 12-hour light-dark cycle, cell division did not display a circadian rhythm. This indicates the absence of circadian gating of cell division in Synechocystis. Instead, we observed a circadian oscillation in the average brightness of the phase contrast in individual Synechocystis cells. To demonstrate how phase-contrast analysis of single cells can be complemented by backscatter analysis of batch cultures, we investigated the wildtype, a deletion mutant known to affect circadian rhythms (∆kaiC3) and complementation strains. We concluded that phase contrast and backscatter likely measured the same rhythmic changes in the refractive index of the cells. The method presented here will advance circadian research by enabling the analysis of circadian rhythms in individual cells without the need for expression of reporter molecules.
Influenza, which causes respiratory tract infections and related complications, poses a major threat to global public health. However, the rapid and accurate detection of influenza viruses in controlling the flu pandemic remains challenging, as current diagnostic methods are static and unable to distinguish between viable and nonviable virus or directly monitor viral replication dynamics. Herein, we report viral luminogenic reporters (VLRs) with chemiluminescence/fluorescence dual-response for non-invasive imaging and urinalysis of the H1N1 virus protease. VLR comprises a bicyclic dioxetane chemiluminophore signaling scaffold caged by a N-acetylneuraminic acid, which further hooks a renal clearable moiety (2-hydroxypropyl)-β-cyclodextrin. VLR achieves a limit of detection of 2.62 CCID50/mL in H1N1 virus detection, which was 10.4-fold and 529.8-fold lower than that of ICA-qPCR and PMA-qPCR assays. After intratracheal administration into H1N1 virus-infected mice, VLR can efficiently accumulate in the lungs and specifically react with neuraminidase to restore its near-infrared chemiluminescent/fluorescent signals for real-time imaging. Leveraging the renal clearance (∼94% ID), VLR allows for remote detection of H1N1 virus infections and monitoring of antiviral therapeutic efficacy through in vitro urinalysis. Therefore, this study highlights a significant advance in addressing the critical gap in dynamic monitoring of virus replication activity and transforming virus-specific probes into urinary reporters.
Common genetic variants contribute to risk for complex human diseases. However, despite thousands of associations, variants modulating disease risk and their functional impact remain largely unknown. This includes SARS-CoV-2 infection, where outcomes range from asymptomatic to fatal. Most genetic risk variants associated with COVID-19 disease, identified through genome wide association studies, are located in the non-coding genome and may function by altering gene expression in disease-relevant cells and tissues. To address this at scale, we tested >4800 severe COVID-19-associated variants to determine the impact of individual variants and variant combinations on regulatory activity using Self-Transcribing Active Regulatory Region sequencing, a massively-parallel reporter assay. Focusing on variants that may have their impact in the lung, in a lung epithelial cell line (A549) we identify 166 variants within active sequences, of which 29 modulate activity allele-specifically. Evaluating variant combinations, we observe both additive and non-additive effects on regulatory activity. We employ state-of-the-art deep learning models to interpret allele-specific variant effects on regulatory activity and endogenous genomic features. Our work provides a set of prioritised severe COVID-19-associated variants that modulate regulatory activity in lung epithelial cells, candidate transcription factors, and candidate target genes with potential to be disease modifying.
Fc gamma receptor IIIA (FCGR3A) encodes CD16a, a key mediator of antibody-dependent cellular cytotoxicity (ADCC) that regulates innate and adaptive immunity, especially in natural killer (NK) cells and monocytes. We generated an FCGR3A-EGFP knock-in human embryonic stem cell (hESC) line via CRISPR/Casn9n. The cell line showed a normal karyotype, maintained expression ofthe pluripotency markers OCT4, SOX2, and NANOG, and retained trilineage differentiation potential. This reporter line enables real-time tracking of FCGR3A expression during immune cell differentiation, serving as a useful tool for studying FCGR3A+ immune cell development and related immune mechanisms.
Accurate and early detection of pneumonia is crucial for effective treatment; however, current diagnostic methods often lack the necessary specificity and sensitivity. Herein, we begin with a comprehensive bioinformatics analysis, identifying neutrophil elastase (NE) as a critical biomarker associated with pneumonia progression. We subsequently develop an NE-responsive probe (NERP), composed of a hemicyanine fluorophore linked to an NE-sensitive peptide, specifically designed for activation in inflamed tissues. To facilitate urinalysis, NERP is further refined into a hydrophilic active targeting responsive probe (ATRPH). ATRPH demonstrates exceptional sensitivity in both in vivo imaging and urine-based detection, with urine analysis offering a noninvasive, early-stage diagnostic option that overcomes the limitations of tissue penetration and low accuracy. In addition, ATRPH is highly effective in drug screening, dosage optimization, and exploring mechanisms of NE production. This biomarker screening and design strategy not only enhances pneumonia diagnosis and treatment via different routes of administration but also has broader potential for other inflammatory lung diseases.
Antibiotic-resistant bacteria are one of the greatest challenges in modern medicine, as multidrug-resistant strains continue to outpace traditional antibiotic development. Unlike conventional antibiotics that promote resistance through selective pressure, quorum sensing-targeted therapies disrupt bacterial communication systems that regulate, among other traits, virulence, biofilm formation, and resistance acquirement through competence without directly killing the bacteria. In this study we examined quorum sensing modulation in Streptococcus mitis, a commensal oral bacterium that has recently been identified as an opportunistic pathogen capable of causing serious infections in immunocompromised individuals. The competence regulon quorum sensing system in S. mitis is controlled by the competence-stimulating peptide (CSP), which activates the histidine kinase receptor ComD to drive quorum sensing-regulated processes. S. mitis strains can be divided into different specificity groups, or pherotypes, based on the CSP they produce and cognate ComD receptor. In this work we set out to define the molecular interactions that drive CSP1/ComD1 binding and lead to ComD1 activation by conducting a systematic structure-activity analysis of the S. mitis-CSP1 sequence. To this end, we synthesized and screened two S. mitis-CSP1 analog libraries: the first, an alanine scan to identify key side-chain residues responsible for activity, and the second, a d-amino acid scan to evaluate the effect of side chain spatial orientation. Then, following the construction of a luminescence-based S. mitis NCTC 8033 quorum sensing reporter strain, we conducted reporter gene bioassays to gain insights into the structure-activity relationship between S. mitis-CSP1 and its cognate ComD1 receptor. Interestingly, our results revealed that the glutamic acid-1-to-alanine analog (S. mitis-CSP1-E1A) had increased potency but lower efficacy, indicating partial agonist effects in S. mitis NCTC 8033, whereas the same glutamic acid-1-to-alanine substitution in CSPs from other Streptococcus species, as well as in a different S. mitis pherotype producing and responding to S. mitis-CSP2, has been reported to produce inhibitory analogs. These results suggest that the ComD1 receptor in S. mitis NCTC 8033 interacts with S. mitis-CSP1 differently than its homologues in other Streptococcus species, highlight the importance of species-specific studies, and caution against overgeneralizing bacterial behavior within a given genus. Overall, our work offers mechanistic insight into S. mitis quorum sensing signaling and lays a foundation for the rational development of CSP-based tools to study and potentially control quorum sensing-regulated processes in streptococci.
Precise knock-in of fluorescent reporters is a powerful tool for studying the dynamic cellular and molecular processes of embryogenesis. However, conventional CRISPR-Cas9 knock-in of large inserts, such as full-length fluorescent proteins, is inefficient. This has limited its application in many emerging model systems, including sea urchins. Here, we overcome this barrier using a transgenic Lytechinus pictus line that constitutively and ubiquitously expresses a large fragment of mNeonGreen (mNG3K 1-10 ). In this line, fluorescence is only reconstituted when CRISPR-mediated knock-in delivers mNG2 11 , the 11th beta strand of the fluorescent protein, to complement the constitutively expressed fragment. Because this strategy requires integrating only the short 11th-strand, together with short homology arms (∼130 nt total), by homology directed repair, it circumvents the size constraints that limit conventional full-length reporter knock-ins using CRISPR. Using this approach, we achieved integration efficiencies of 14-22%, roughly an order of magnitude higher than those obtained with full-length fluorescent protein knock-ins. This provides a streamlined, scalable method for endogenous protein visualization in echinoderm embryos and a valuable resource for studying gene function, morphogenesis, and toxicant response in this classic developmental model.
Red blood cell (RBC) transfusions are essential for treating various medical conditions, but global demand is difficult to meet due to a dwindling donor pool and compatibility issues. Pluripotent stem cells (PSCs) offer a promising alternative of blood dependent on volunteer donors for RBC production, and dogs serve as an excellent model for translational research due to their physiological and genetic similarities to humans. Canine induced pluripotent stem cells (ciPSCs) were differentiated toward hematopoietic and erythroid lineages. Differentiated cells were evaluated for hematopoietic marker expression, hemoglobinization, colony-forming capacity, enucleation, and hemoglobin gene expression. Glycophorin A (GYPA)-enhanced green fluorescent protein (EGFP) reporter ciPSC lines were generated using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated genome editing to visualize GYPA expression during differentiation. This study introduces a protocol for RBC differentiation using ciPSCs. We achieved generation of hemoglobinized RBCs, progressing through polychromatic and orthochromatic erythroblast-like stages. CiPSC-derived hematopoietic cells/RBCs were confirmed to have immature characteristics as determined by limited colony-forming capacities, low enucleation, and embryonic and fetal hemoglobin gene expression. Additionally, we created GYPA-EGFP reporter ciPSC lines using CRISPR-Cas9-mediated genome editing, enabling real-time visualization of GYPA expression. This innovation confirmed GYPA as a viable surface marker for ciPSC-derived RBCs. Our findings mark an initial step toward establishing a canine PSC-based erythroid differentiation system, providing a foundation for future improvements and exploration of applications for canine PSC-derived RBCs.
In this investigation, we examined the functional mechanism of the transcription factor zinc finger protein 695 (ZNF695) and its target gene chromobox protein homolog 8 (CBX8) in colorectal cancer (CRC) migration and invasion. HCT-116 and LOVO cell lines were used to establish cell models with knocked-down ZNF695 and knocked-down or over-expressed CBX8. To comprehensively evaluate the functional contributions of ZNF695 and CBX8 to cellular phenotypes, we employed CCK-8, wound-healing, and Transwell assays to evaluate cell proliferation, migration, and invasion, respectively. To assess the impact of ZNF695 on tumor progression, we generated a xenograft model utilizing nude mice. A FLAG-ZNF695 expression plasmid was constructed, and ChIP-seq experiments were performed. By integrating mRNA sequencing data following ZNF695 knockdown with highly expressed genes in CRC from the TCGA database, CBX8 was identified as a putative downstream target of ZNF695. We employed a dual-luciferase reporter assay to validate the specific binding affinity of ZNF695 toward the CBX8 promoter region. To elucidate the specific biological cascades modulated by ZNF695 and CBX8, we conducted a comprehensive pathway enrichment analysis. Rescue experiments were conducted to determine whether the ZNF695/CBX8 regulatory axis upregulates the expression of the Wnt signaling pathway downstream targets, AXIN2 and CCND1. Both in vitro assays and in vivo models confirmed that silencing ZNF695 dramatically suppresses CRC cell proliferation, migration, and invasion, while concurrently impeding tumor progression. ChIP-seq coupled with dual-luciferase reporter assays substantiated the direct binding of ZNF695 to the CBX8 promoter. Furthermore, CBX8 depletion significantly attenuated the migratory and invasive phenotypes of CRC cells. Restoring CBX8 expression effectively rescued the migratory and invasive deficits in CRC cells induced by ZNF695 silencing. Re-expression of CBX8 in ZNF695-silenced cells restored Wnt/β-catenin signaling activity, accompanied by increased expression of AXIN2 and CCND1. ZNF695 promotes CRC progression by transcriptionally activating CBX8 and subsequently enhancing Wnt/β-catenin signaling, thereby promoting tumor cell proliferation, migration, and invasion.
Understanding disease-associated metabolic reprogramming requires comprehensive interrogation of the chemically diverse metabolome. However, conventional liquid chromatography-mass spectrometry (LC-MS) workflows analyze metabolites in a largely non-discriminatory manner, resulting in systematic underrepresentation of specific functional and reactivity classes due to heterogeneous ionization efficiencies and matrix interference. Here, we report a chemoselective metabolomics strategy based on a modular reactivity-encoding platform (MREP) that enables functional group-resolved stratification of complex metabolomes. Four orthogonally designed alkyne-tagged probes selectively derivatize carboxyl, carbonyl, amine, and thiol functionalities under compatible conditions. The encoded metabolites are subsequently immobilized via azide-alkyne cycloaddition onto a unified solid-phase capture resin, which simultaneously removes matrix components and installs a diagnostic reporter module. This integrated encoding-capture architecture achieves high reaction orthogonality, near-quantitative conversion, and robust quantitative performance across structurally diverse metabolites. The resulting triazole derivatives exhibit markedly enhanced ionization efficiencies and generate a universal reporter-ion, enabling confident submetabolome classification and reconstruction. Application to serum and liver tissues from mice substantially expands the detectable chemical space, yielding 7 208 features and 1 573 annotated metabolites across four functional group-defined layers. Collectively, this work establishes the MREP framework as a versatile platform for reactivity-resolved interrogation of complex small-molecule systems.
Fluorescent reporters are powerful tools to reveal intercellular heterogeneity among proliferating cells. However, there are few tools to analyze differences among quiescent (G0) cells, though such differences are relevant for development, tissue maintenance, and cancer cell behavior. Quiescence heterogeneity, also known as quiescence depth, typically correlates with time after cell cycle arrest, yet directly measuring cell age is not feasible for all cell types or most tissues. Here, we describe ELDR-Glo, a genetically-encoded fluorescent biosensor that estimates relative cell age, i.e., time since the last cell cycle. The biosensor integrates replication-coupled degradation in S phase with a slow-maturing mCherry and a normalization module. We demonstrate that ELDR-Glo signal correlates with true cell age by both live-cell imaging and in fixed cells. ELDR-Glo distinguishes early and late G0 cells and functions as a relative quiescence depth reporter in situ. The biosensor is compatible with multiplexed immunofluorescence and flow cytometry. ELDR-Glo provides a unique and scalable tool to investigate cell proliferation control.
This study aimed to investigate the role and mechanism of T-box transcription factor 20 (TBX20) in doxorubicin resistance in breast cancer cells. RNA-seq data from breast cancer samples in the TCGA database were analyzed. Lentiviral vectors were used to establish TBX20 overexpression and silencing models in MCF-7 and MDA-MB-231 cells. Gene and protein expression were detected by qPCR and Western blot, respectively. Cell viability and the half-maximal inhibitory concentration of doxorubicin were measured using the CCK-8 assay. Apoptosis, migration, and invasion were analyzed by flow cytometry, wound healing assay, and Transwell assay. Mitophagy levels were assessed via immunofluorescence staining and western blotting. ChIP and dual-luciferase reporter assays were performed to validate the transcriptional regulation of ABCC1 by TBX20. Results showed that TCGA data analysis revealed a high expression of TBX20 in breast cancer tissues, which was positively correlated with ABCC1 expression. In MCF-7 and MDA-MB-231 cells, TBX20 overexpression significantly enhanced cell proliferation, migration, invasion, and resistance to doxorubicin, while suppressing the expression of mitophagy-related proteins LC3-II/LC3-I, PINK1, and BNIP3. ChIP and dual-luciferase reporter assays confirmed that TBX20 directly binds to and activates the ABCC1 promoter. Silencing of ABCC1 or restoration of mitophagy by CCCP reversed TBX20 overexpression‑induced doxorubicin resistance. TBX20 enhances the resistance of breast cancer cells to doxorubicin by transcriptionally upregulating ABCC1 and is correlated with the suppression of mitophagy.
The quality of in vitro maturation (IVM) is a major factor limiting the efficiency of in vitro fertilization (IVF) and early embryonic development in livestock. This study aimed to validate, in sheep oocytes, the functional relevance of two conserved core miRNA-mRNA interactions, miRNA-504 (miR-504)/pyruvate dehydrogenase alpha 1 (PDHA1) and miRNA-500 (miR-500)/Egl nine homolog 2 (EGLN2), previously identified in porcine oocytes. Using sheep cumulus-oocyte complexes (COCs) as the experimental model, we combined bioinformatic analysis, dual-luciferase reporter assays, transfection of miRNA mimics and inhibitors, quantitative reverse transcription PCR (qRT-PCR), Western blotting (WB), fluorescence staining, IVM, IVF, and in vitro culture (IVC). Through these approaches, we systematically evaluated the effects of the two regulatory axes on sheep oocyte maturation quality, redox status, mitochondrial function, and subsequent early embryonic development, and further assessed the effects of combined intervention. The results showed that miR-504 and miR-500 were highly conserved among multiple mammalian species, with highly consistent seed sequences. Dual-luciferase assays confirmed that miR-504 and miR-500 directly targeted PDHA1 and EGLN2, respectively. Functional analyses showed that overexpression of either miR-504 or miR-500 suppressed cumulus expansion and increased apoptosis, with apoptosis rates increasing from 14.26% to 15.53% in the corresponding control groups to 18.08% and 19.46%, respectively. At the same time, intracellular reactive oxygen species (ROS) levels increased significantly, whereas glutathione (GSH) content and mitochondrial membrane potential decreased significantly. The expression of key developmental factors, including growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and cyclin-dependent kinase 1 (CDK1), also declined. These findings indicate that both pathways impair developmental competence by disrupting cytoplasmic maturation. Developmental phenotype analysis further showed that inhibition of miR-504 or miR-500 increased cleavage rate, morula rate, and blastocyst rate. Blastocyst cell numbers were significantly higher in the miR-504 inhibitor and miR-500 inhibitor groups than in the control group (p < 0.05). Molecular analyses showed that miR-504 negatively regulated PDHA1 and its associated energy metabolic pathway, whereas miR-500 negatively regulated the EGLN2/hypoxia-inducible factor-1α (HIF-1α) pathway. In addition, the combined treatment group receiving both miR-504 inhibitor and miR-500 inhibitor outperformed either single-treatment group in morula formation, blastocyst formation, and blastocyst cell number (p < 0.05), indicating an enhanced effect of combined inhibition in sheep oocytes. In summary, this study supports cross-species conservation at the level of the core miRNA-mRNA interactions of miR-504/PDHA1 and miR-500/EGLN2 and supports their functional relevance in sheep oocytes. These findings provide new evidence for understanding the molecular mechanisms that regulate mammalian oocyte quality and offer a theoretical basis for improving in vitro embryo production (IVP) in sheep.
Taste buds (TBs) are the fundamental units of taste perception and are distributed throughout the oral cavity, including the tongue, soft palate, epiglottis, pharynx, and retromolar pad. Despite their widespread presence, a complete and systematic understanding of TB distribution and functional organization remains incomplete. In this study, we focus on a small population of TBs located in the mandibular sublingual caruncle of mice, adjacent to the openings of the Wharton's ducts, a site previously noted in anatomical and physiological studies that has not been comprehensively characterized. TBs of the sublingual caruncle exhibit canonical morphology and cellular composition, comprising type I (NTPDase2 +), type II (Trpm5 +), and type III (Car4 +) cells, as confirmed by transgenic reporter lines and immunofluorescence staining. Molecular analysis revealed the expression of key taste transduction components, including Gnat3, Trpm5, and Plcb2, as well as 26 of 35 bitter taste receptors. Immunofluorescence confirmed TB innervation by βIII-tubulin-positive nerve fibers, including P2X3-positive sensory afferents, Synapsin-1-positive presynaptic terminals, and CGRP-positive peptidergic endings, indicating integration into the peripheral gustatory circuitry. Retrograde neuronal tracing (Dil/DiO) further demonstrated that nerve fibers associated with sublingual caruncle TBs project specifically to the geniculate but not the petrosal ganglion, supporting their attribution to the facial gustatory pathway. Functionally, localized sucrose stimulation of the sublingual caruncle elicited cFos activation in the nucleus of the solitary tract, indicating central connectivity. Furthermore, tastants representing all 5 basic taste qualities (sweet, sour, bitter, umami, and salty) elicited measurable saliva secretion, with bitter-, sweet-, and umami-induced salivation reduced in transgenic mouse models with impaired gustatory function. Together, these findings establish the sublingual caruncle as a functional gustatory site involved in taste-evoked salivary reflexes, expanding current understanding of taste system organization and its coordination with salivary function.
The oriental fruit fly Bactrocera dorsalis is a globally invasive pest with increasing insecticide resistance that threatens sustainable crop production. ATP-binding cassette (ABC) transporters are key mediators of xenobiotic detoxification, but the molecular and regulatory basis of ABCC-type transporters in this species remains unclear. Here, we present the first systematic reannotation and functional analysis of the ABCC gene family in B. dorsalis and identify a novel post-transcriptional regulatory pathway involving miR-980. Using updated genomic and transcriptomic resources, we accurately annotated eight BdABCC genes with conserved nucleotide-binding (NBD) and transmembrane domains (TMD) and strong phylogenetic conservation with dipteran homologs. Spatiotemporal expression profiling showed constitutive expression across development, elevated transcription in the midgut, Malpighian tubules and fat body, and transcriptional responsiveness of several members, particularly BdABCC1, BdABCC2, BdABCC4, and BdABCC6, to multiple insecticides. RNA interference and MK-571 inhibition demonstrated that BdABCC1 contributes to avermectin tolerance. Furthermore, luciferase reporter assays, RNA pull-down and fluorescence in situ hybridization confirmed that miR-980 directly targets the BdABCC1 coding sequence, with miR-980 overexpression suppressing BdABCC1 and increasing avermectin-induced mortality. Together, these findings identify a previously uncharacterized miR-980-BdABCC1 regulatory axis that modulates avermectin tolerance and highlights post-transcriptional regulation of ABC transporters as a potential molecular target for future RNAi-based pest management strategies.
Implementation science (IS) plays a critical role in translating research into real-world health outcomes. Few studies have evaluated models that provide technical assistance and other resources to build IS capacity. The Coordinating and Capacity-Building Hubs to Enhance the Science of HIV Implementation Research (CHESHIRE) network supports US-based HIV research awardees participating in the Ending the HIV Epidemic in the US (EHE) initiative. The objective of this study is to describe the evaluation protocol for CHESHIRE and assess the effect of the implementation of hub technical assistance activities on EHE-funded research team outcomes, including IS competencies, scientific collaboration, and research productivity. This protocol describes a mixed methods evaluation using qualitative interviews with EHE project leads (n=36); social network analysis of CHESHIRE-affiliated researchers and hub members (n=265); and secondary data analysis of National Institutes of Health (NIH) RePORTER, PubMed, and NIH Implementation Science Coordination Initiative EHE Project Final Progress Report Survey data (n=248). We will use descriptive analyses, network metrics, and thematic coding to describe outcomes following CHESHIRE implementation, including IS competencies, interinstitutional partnerships, and research productivity. CHESHIRE coordinating center and hub activities were funded through NIH Center for AIDS Research and AIDS Research Center supplements beginning in 2019, with hubs funded between 2019 and 2024. Available survey data include EHE projects that completed the EHE Project Final Progress Report Survey between August 2021 and February 2025. As of January 2026, we have completed enrollment, with 265 network members in the final recruiting list. Data abstraction and analysis of the evaluation components are ongoing. Publication of findings is anticipated for December 2026. Evaluation findings will be disseminated after completion of data analysis. The findings will provide insights on whether structured IS support through CHESHIRE increases researchers' IS competencies, interinstitutional partnerships, and research productivity. This evaluation will provide empirical evidence to guide the development and optimization of technical assistance hubs in public health research and inform their future evaluation of hub-based IS capacity-building models. Findings will inform strategies to optimize IS capacity building and accelerate the translation of evidence-based interventions into practice, especially in HIV prevention and treatment. DERR1-10.2196/91374.
Zebrafish combines the power of genetics and unparalleled in vivo imaging for investigating the dynamics of vertebrate hematopoietic development. Across species, the transcription factor Runx1 is essential for definitive hematopoiesis. We generated a zebrafish runx1-2A-creERT2 CRISPR knock-in for tamoxifen-regulated Cre recombinase Runx1 lineage tracing and characterized its activity using the ubi:Switch recombinase-dependent fluorescence reporter, microscopic live imaging and flow cytometry. Tamoxifen treatment beginning at gastrula stage labeled all expected Runx1 lineages in the early embryo, including neuroectodermal olfactory placode and Rohan-Beard neurons, primitive hematopoietic blood cells, and nascent hematopoietic stem and progenitor cells (HSPCs) in the dorsal aorta. Runx1 HSPCs colonized the larval caudal hematopoietic tissue and thymus from three to five days of development. Timed tamoxifen induction of Cre activity allowed separation of Runx1 primitive hematopoiesis from definitive HSPC emergence and larval stem cell niche colonization. Flow cytometry of kidney marrow and peripheral blood from adults treated with tamoxifen at gastrula stage revealed Runx1 embryonic hematopoietic cells contributed to adult hematopoietic precursors, myeloid, lymphoid, and peripheral blood lineages. Labeling of all blood lineages was also effective by tamoxifen treatment of 5-month-old adults. The zebrafish runx1-2A-creERT2 line provides a powerful tool for precise spatial and temporal analysis of Runx1 progenitor mechanisms in developmental and adult hematopoiesis. zebrafish endogenous runx1-2A-creERT2 provides inducible Cre recombinase genetic analysis in all runx1 neuromesodermal and blood lineages zebrafish runx1-2A-creERT2 line enables in vivo spatial and temporal analysis of embryonic and adult hematopoiesis.
Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide. Understanding the complex molecular networks that underlie this aggressive behavior is critical for developing novel diagnostic and therapeutic strategies. This study aimed to identify key molecular regulators of CRC progression by integrating Weighted Gene Co-expression Network Analysis (WGCNA) with machine learning algorithms. Hub genes were initially identified by intersecting genes from the most significant module with CRC-related and glycolysis-related targets from the GeneCards database, as well as upregulated differentially expressed genes (DEGs) from the GSE113513 dataset. Lasso regression and random forest (RF) algorithms were employed to screen for key genes from this intersection. The expression of the identified key gene was validated using quantitative real-time PCR (qRT-PCR) and Western blotting. Functional assays, including Cell Counting Kit-8 (CCK-8), colony formation, Transwell invasion, flow cytometry, and metabolic analyses, were conducted to analyze the malignant behaviors of CRC cells. The regulatory relationship between cyclin dependent kinase 1 (CDK1) and transcription factor AP-4 (TFAP4) was validated through chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays. A xenograft mouse model was used to evaluate the effect of TFAP4 knockdown on the malignant progression of CRC cells in vivo. WGCNA and machine learning analyses identified three key genes: MET, MYC, and CDK1. CDK1 was selected for further investigation and found to be significantly upregulated in CRC tissues and cell lines. Functionally, CDK1 knockdown markedly inhibited CRC cell proliferation, invasion, and glycolysis while promoting apoptosis. Mechanistically, the transcription factor TFAP4 was identified as an upstream regulator that directly activated CDK1 transcription. Moreover, CDK1 and TFAP4 expression were associated with metastatic stage. TFAP4 exerted its oncogenic effects by positively regulating CDK1. Furthermore, silencing TFAP4 significantly suppressed tumor growth in vivo. This study establishes the TFAP4-CDK1 axis as a critical driver of malignant progression in CRC. Targeting this pathway could lead to the development of novel interventions for CRC.