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.
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.
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.
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.
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.
Ischemic stroke is the most prevalent form of stroke worldwide and remains a major cause of long-term disability. Impaired autophagic flux is a critical pathological mechanism that worsens neuronal injury after ischemia. This study aimed to elucidate the role of phosphatidylinositol-5-phosphate 4-kinase type II alpha (PIP4K2A) and its regulation of autophagy in cerebral ischemia/reperfusion (I/R) injury. Exploratory TMT-based serum proteomics identified PIP4K2Aas an elevated candidate protein in patients with acute ischemic stroke (AIS). Whole-blood RT-qPCR validation showed increased PIP4K2A mRNA in AIS patients and an association between higher PIP4K2A expression and lower NIHSS scores, although the small clinical cohort and peripheral sampling design preclude causal or tissue-origin conclusions. Using a transient middle cerebral artery occlusion (tMCAO) mouse model and a primary neuronal oxygen-glucose deprivation/reperfusion (OGD/R) model, we found that I/R injury markedly upregulated PIP4K2A expression in ischemic brain tissue and primary neurons. AAV-mediated PIP4K2A overexpression in vivo alleviated ischemic brain injury, preserved neuronal survival, reduced infarct volume, and improved long-term cognitive and motor functions, whereas PIP4K2A knockdown exacerbated these outcomes. In vitro, lentiviral-mediated PIP4K2A overexpression improved neuronal viability after OGD/R. Mechanistically, RNA-seq, co-immunoprecipitation, and mCherry-EGFP-LC3 tandem reporter analyses showed that PIP4K2A overexpression was associated with reduced TRIB3 mRNA and protein levels, decreased abundance of the stress-induced TRIB3-p62 complex, improved autophagic flux, and reduced autophagosomal accumulation. Concurrently, PIP4K2A-associated TRIB3 reduction was accompanied by enhanced AKT/mTOR signaling. These findings identify PIP4K2A as an endogenous protective regulator in cerebral ischemia/reperfusion injury and suggest that thePIP4K2A/TRIB3/p62 axis may represent a potential therapeutic target for ischemic stroke.
Whole-cell biosensors provide a cost-effective and sensitive approach for real-time monitoring of toxic metals in environmental samples. In this study, a bacterial whole-cell biosensor was engineered using Escherichia coli BL21(DE3) by integrating the cadC regulatory gene from Bacillus megaterium TWSL_4 with a green fluorescent protein (GFP) reporter to enable fluorescence-based detection of heavy metals. The biosensor gene cassette (Pcad + cadC + gfp) was first cloned into the pUC19 vector and then subcloned into the pET28a(+) expression vector to generate the recombinant plasmid pETCG28. Functional characterization showed that the engineered strain E. coli BL21/pETCG28 exhibited enhanced tolerance to heavy metals, sustaining growth at Pb²⁺ concentrations up to 1600 ppm, Cd²⁺ up to 200 ppm, and Zn²⁺ up to 60 ppm, significantly higher than the wild-type strain. Fluorescence analyses demonstrated strong concentration-dependent responses to heavy metal exposure. Corrected total cell fluorescence increased nearly fourfold between 1 ppb and 10 ppb of Pb²⁺ (R² = 0.95, p < 0.0001). Cd²⁺ exposure produced an approximately threefold increase (R² = 0.96, p < 0.0001), while Zn²⁺ generated a moderate twofold response (R² = 0.94, p < 0.0001). Optimal biosensor performance occurred at pH 7.0 and 37°C, demonstrating potential for portable environmental monitoring applications.
The biological oxidation of ammonia, the first step of nitrification, is central to biological water purification processes for nitrogen removal. For drinking water treatment, particularly sourced from groundwater, low concentrations of available copper often limit the efficiency of nitrification. Copper dosing both enhances nitrification and affects the composition of the nitrifying microbial community. The mechanisms underlying the effect of copper on nitrifying community composition, ammonia oxidation, and subsequent nitrogen removal processes remain unknown. The objective of this study was to confirm the effects of copper availability on the relative abundance of complete (comammox) and canonical ammonia-oxidizing bacteria (AOB) in nitrifying communities within the drinking water treatment plant and to determine differences in their copper transport mechanisms. Comparative metagenomic analysis revealed that, unlike most AOB, many comammox Nitrospira encode PcoB/CopB-type high-affinity copper uptake systems, indicating that they are more competitive in low-copper environments. This niche adaptation was confirmed in laboratory-scale bioreactors, which showed that comammox Nitrospira became dominant under copper-limited conditions, while AOB dominated at high copper concentrations. Furthermore, specific detection of comammox amoA mRNA by catalyzed reporter deposition-fluorescent in situ hybridization confirmed that the transcriptional activity of comammox Nitrospira was higher compared to AOB under copper limitation. Thus, these results suggest that copper availability may play an important role in shaping the dominant ammonia-oxidizing bacterial guild, with potential implications for engineered water treatment processes.
Pediatric anal fistula is a clinically distinct condition. Postoperative recurrence remains a major therapeutic challenge, as the underlying molecular mechanisms are poorly understood. To investigate the potential role of the SIRPG-AS1/miR-3692-5p/STAT1 axis in pediatric anal fistula progression and recurrence through bioinformatics analysis and experimental validation. Bioinformatics analysis of the GSE157020 dataset identified differentially expressed long non-coding RNAs (lncRNAs). The downstream miRNAs and target genes were predicted using multiple databases. The regulatory axis was validated using RIP, RNA pull-down, and dual-luciferase reporter assays. Clinical relevance was assessed in serum from 80 primary and 25 recurrent pediatric idiopathic anal fistula cases, compared with 80 healthy controls. Functional studies were performed in TGF-β-treated CCD-18Co cells with genetic manipulations. Analysis of database GSE157020, SIRPG-AS1 was found to be significantly upregulated in anal fistula tissues. The SIRPG-AS1/miR-3692-5p/STAT1 axis was identified, with SIRPG-AS1 regulating STAT1 expression by competitively binding to miR-3692-5p. Clinical sample analysis revealed significant changes in the expression levels of SIRPG-AS1, miR-3692-5p, and STAT1 (as assessed in serum) in pediatric patients with anal fistula, correlating with disease severity and recurrence. In vitro experiments showed that the SIRPG-AS1/miR-3692-5p/STAT1 axis was involved in TGF-β-induced fibrosis in CCD-18Co cells. Our study suggests that the SIRPG-AS1/miR-3692-5p/STAT1 axis may play a role in pediatric anal fistula progression and recurrence. Further investigation into this axis could provide additional insights into the mechanisms underlying pediatric anal fistula.
In this study, we developed an aptamer-mediated surface-enhanced Raman scattering (SERS)-coupled immunochromatographic assay (ICA) for the quantitative detection of pyrethroids. For this approach, core-shell gold nanoparticles (Au@Au NPs) were used as SERS probes by conjugating with aptamers, and fluorescent Raman reporter molecules (MPBN) were incorporated to yield stable, reproducible spectral signals. Leveraging the aptamers' specific recognition of the target analyte, Raman-labeled Au@Au NPs could be effectively captured by the test line of the immunochromatographic strip, thereby enabling the quantitative analysis of fenvalerate-with a limit of detection (LOD) as low as 0.4 ppb. Comparative experiments revealed that the detection sensitivity of this SERS-ICA method was 476 times higher than that of conventional lateral flow assays (LFA), showcasing a remarkable sensitivity advantage. To validate its practicality, spiked recovery experiments were conducted on three real samples, namely welsh onions, cowpeas, and kidney beans. The results showed that the spiked recoveries ranged from 78.3% to 112.5%, with relative standard deviations (RSD) between 1.7% and 20.1%. This fully confirms that the method possesses favorable specificity and accuracy, making it suitable for real-sample detection. This detection platform offers a new technical route for the rapid on-site monitoring of trace contaminants in food safety and environmental surveillance, holding broad application prospects.
Mitochondria are dynamic organelles essential for neuronal survival and synaptic function, and their dysfunction is a key consequence of excitotoxicity following traumatic brain injury (TBI). While intercellular mitochondrial transfer and exogenous mitochondrial transplantation have emerged as mechanisms to restore cellular bioenergetics, its in vivo relevance in the central nervous system remains incompletely understood. Here, we used astrocyte and neuron-specific mitochondrial reporters (GFP or Dendra2) in mice to assess cell-type-specific mitochondrial morphology, bioenergetics, and transfer 24hrs after TBI. Neurons exhibited marked mitochondrial dysfunction, including altered morphology and reduced bioenergetic capacity across somatic, synaptic, and non-neuronal fractions. In contrast, astrocytic mitochondria showed morphological changes but preserved bioenergetic function. Concomitantly, astrocyte-to-neuron mitochondrial transfer was significantly increased following injury, although transfer to synapses remained limited. Single-cell RNA sequencing of astrocytes revealed upregulation of genes involved in extracellular vesicle (EV) biogenesis and mitochondrial translation following injury compared to controls. In vitro co-culture studies confirmed that astrocytes transfer mitochondria to neurons via EVs containing mitochondria (EV-mito). Isolated EV-mito from astrocyte-conditioned media improves neuronal mitochondrial function under NMDA (N-methyl-D-aspartate) induced excitotoxic conditions. Together, these findings demonstrate that neuronal mitochondrial dysfunction drives astrocyte-mediated mitochondrial transfer as an adaptive neuroprotective response after TBI. This process preserves neuronal bioenergetics in the soma and neurites but not at synapses, highlighting both its therapeutic potential and spatial limitations.
To systematically characterize the post-marketing safety signals of isotretinoin using real-world data from the U.S. FDA Adverse Event Reporting System (FAERS), with independent external validation in the European EudraVigilance (EV) database. Adverse event (AE) reports in FAERS from 2004Q1 to 2024Q3 were analyzed. Signal detection was conducted using four complementary disproportionality algorithms: reporting odds ratio (ROR), proportional reporting ratio (PRR), Bayesian confidence propagation neural network (BCPNN), and the multi-item gamma Poisson shrinker (MGPS). Signals concurrently detected by all four methods were defined as robust. Key findings were subsequently examined in EV as an external reference. Among 50,519 patients contributing 142,160 isotretinoin-associated AE reports, 469 statistically robust signals were identified, spanning 25 system organ classes (SOCs). Signals were most concentrated in psychiatric disorders (75, 15.99%), gastrointestinal disorders (58, 12.37%), and congenital, familial, and genetic disorders (50, 10.66%). The strongest association was observed for inflammatory bowel disease (IBD; ROR = 579.14); however, temporal clustering and reporter-type profiling suggested substantial stimulated reporting, potentially driven by litigation, warranting cautious interpretation. Several high-ranking signals were not described in current product labeling, including nasal vestibulitis, hypertrophic anal papilla, and SAPHO syndrome. Pregnancy-related signals were prominent, with unintended pregnancy showing a strong signal (ROR = 91.39). External validation in EV demonstrated high concordance, supporting the robustness and reproducibility of the findings. Isotretinoin is associated with a broad spectrum of pharmacovigilance signals, with disproportionate representation of psychiatric, gastrointestinal, and pregnancy-related events. While multiple previously unlabelled signals emerged, their clinical relevance remains to be established. These findings underscore the need for strengthened clinical monitoring, rigorous pregnancy prevention strategies, and careful interpretation of spontaneous reporting data.
To identify, map, and characterize a posterior ocular lymphatic outflow (POLO) pathway from the suprachoroidal space and assess its drainage dynamics and cervical nodal uptake using time-resolved imaging. Albumin-based tracers (galbumin and CF770/BSA) were delivered into the suprachoroidal space of adult mice under general anesthesia. In vivo imaging included magnetic resonance imaging (MRI) and near-infrared fluorescence imaging using confocal scanning laser ophthalmoscopy (cSLO) of the ocular surface and orbital soft tissues. Ex vivo assessments included in situ head and neck fluorescence imaging 20 minutes after tracer injection and hyperspectral microscopy of ocular and orbital sections. Lymphatic identity was assessed by immunofluorescence staining (podoplanin and vascular endothelial growth factor receptor-3 [VEGFR-3]) in Prox1-tdTomato reporter mice. Following suprachoroidal tracer injection, in vivo MRI and cSLO fluorescence imaging showed preferential drainage toward the nasal orbit. Hyperspectral microscopy showed higher tracer signal in the ipsilateral sclera and orbit than contralateral tissues, and in situ fluorescence imaging detected near-infrared tracer in the ipsilateral cervical lymph node at 20 minutes. Confocal microscopy of the choroid showed co-localization of podoplanin with VEGFR-3 and Prox1 and demonstrated tracer within podoplanin-positive choroidal lymphatic vessels, consistent with a posterior drainage route from the suprachoroidal space to cervical lymph nodes. This study identifies a previously unrecognized POLO pathway and expands ocular drainage physiology beyond the anterior segment to include a posterior lymphatic route through choroidal lymphatic vessels. Identification of a POLO pathway reveals a measurable route for fluid clearance, intraocular pressure regulation, and drug delivery, offering new strategies to preserve vision in retinal and posterior segment diseases characterized by fluid accumulation, impaired clearance, or inflammation.
Human pluripotent stem cell (hPSC) manufacturing workflows frequently rely on suspension aggregation, yet inter-line and batch-to-batch variability in aggregate formation can compromise process consistency and downstream differentiation performance. We evaluated whether a short exposure to HA-100, a small-molecule inhibitor of protein kinase A and protein kinase C signaling, could be used as an upstream process intervention to improve aggregate uniformity without compromising hPSC identity or developmental competence. Nine hPSC lines, including human embryonic stem cell and induced pluripotent stem cell lines, were examined in suspension culture. HA-100 was applied during the first 24 h of aggregation. Aggregate morphology and size distribution were assessed across lines. To investigate the cellular basis of this effect, we generated an mCherry-TJP1 reporter hESC line, which enabled live visualization of junction dynamics, including responses under calcium-depleted conditions and recovery of transepithelial electrical resistance. HA-100 treatment promoted more compact and spherical aggregates, increased aggregate size into a narrower range across lines, and reduced overall variability relative to medium alone. Across the nine-line panel, HA-100-treated aggregates fell within an empirically definesd size range of 25.37-33.95 x 10^-4 mm^3 after 24 h of suspension culture, providing a practical benchmark for process monitoring. In calcium-depleted conditions, HA-100 delayed disruption of intercellular contacts and accelerated recovery of transepithelial electrical resistance, consistent with improved junctional resilience. Transient exposure to HA-100 did not abolish pluripotency marker expression or tri-lineage differentiation capacity. These data support HA-100 as a practical upstream intervention to reduce aggregate heterogeneity in suspension hPSC cultures and improve reproducibility in manufacturing-oriented workflows requiring consistent aggregation.
Following the ubiquitous autotrophic ammonia-oxidizing archaea (AOA), heterotrophic representatives of the marine Nitrososphaerota (HMN) form the second most abundant group within this archaeal phylum. However, their eco-evolutionary strategies remain poorly understood. Previous studies have reported a consistent co-occurrence of HMN with marine AOA (MAOA), prompting a detailed investigation into their potential interaction. Through large-scale (meta)genomic and metatranscriptomic analyses, we reveal that HMN possess ultra-streamlined genomes and globally co-occur with marine AOA. The absence of most B vitamin biosynthesis pathways, incomplete citrate cycle and glycolysis, along with the essential requirement for exogenous amino acids, suggest their potential metabolic dependency on AOA. Meanwhile, catalyzed reporter deposition fluorescence in situ hybridization supports a close physical association between HMN and AOA. The nearly synchronous origins of HMN and AOA after oxygen rise, coupled with HMN's dispersive microhabitats (evidenced by dense, shallow subclades) and extensive horizontal gene transfer between these groups, further support their close relationship-although HMN likely acquired heterotrophic capabilities from bacteria. This study reveals a previously unrecognized association between HMN and AOA, implying a tight coupling between autotrophic and heterotrophic processes in deep-sea habitats.