Zebrafish Larvae Microinjection and Automated Fluorescence Microscopy for Studying Klebsiella pneumoniae Infection and the Host Immune Response.

Studying host-pathogen interactions is essential for understanding infectious diseases and developing possible treatments, especially for priority pathogens with increased virulence and antibiotic resistance, such as Klebsiella pneumoniae. Over time, this subject has been approached from different perspectives, often using mammal host models and invasive endpoint measurements (e.g., sacrifice and organ extraction). However, taking advantage of technological advances, it is now possible to follow the infective process by noninvasive visualization in real time, using optically amenable surrogate hosts. In this line, this chapter describes a live-cell imaging approach to monitor the interaction of K. pneumoniae and potentially other bacterial pathogens with zebrafish larvae in vivo. This methodology is based on the microinjection of fluorescent bacteria into the otic vesicle, followed by time-lapse observation by automated fluorescence microscopy with environmental control, monitoring the dynamics of immune cell recruitment, bacterial load, and larvae survival.

Memory effects of transcription regulator-DNA interactions in bacteria.

Memory effect refers to the phenomenon where past events influence a system's current and future states or behaviors. In biology, memory effects often arise from intra- or intermolecular interactions, leading to temporally correlated behaviors. Single-molecule studies have shown that enzymes and DNA-binding proteins can exhibit time-correlated behaviors of their activity. While memory effects are well documented and studied in vitro, no such examples exist in cells to our knowledge. Combining single-molecule tracking (SMT) and single-cell protein quantitation, we find in living Escherichia coli cells distinct temporal correlations in the binding/unbinding events on DNA by MerR- and Fur-family metalloregulators, manifesting as memory effects with timescales of ~1 s. These memory effects persist irrespective of the type of the metalloregulators or their metallation states. Moreover, these temporal correlations of metalloregulator-DNA interactions are associated with spatial confinements of the metalloregulators near their DNA binding sites, suggesting microdomains of ~100 nm in size that possibly result from the spatial organizations of the bacterial chromosome without the involvement of membranes. These microdomains likely facilitate repeated binding events, enhancing regulator-DNA contact frequency and potentially gene regulation efficiency. These findings provide unique insights into the spatiotemporal dynamics of protein-DNA interactions in bacterial cells, introducing the concept of microdomains as a crucial player in memory effect-driven gene regulation.

Pyrene Based Schiff Base for Cascade Fluorescent "On-Off" Detection of Aluminium(III) and Fluoride ions and its Applications in Live Cells Imaging.

An easy-to-prepare pyrene-based Schiff base PNZ was synthesized by condensing equimolar amount of 1-pyrenebutyric hydrazide with 2-hydroxy-naphthaldehyde, and employed as a fluorescent chemosensor for in-situ cascade detection of aluminium (Al3+) and fluoride (F-) ions. In DMSO:H2O (1:1, v/v), the weakly emissive PNZ showed a significant fluorescence enhancement at 455 nm selectively upon the addition of Al3+ due to the complexation-induced formation of a pyrene excimer. Schiff base PNZ and Al3+ formed a complex in 2:1 binding ratio with the estimated binding constants of K1:1 = 9826.01 M-1 and K2:1 = 3188.49 M-1. The sensing mechanism was explored by performing quantum mechanical calculations and 1H NMR titration of PNZ with Al3+. The in-situ formed PNZ-Al3+ complex species enabled the fluorescent turn-off detection of F-. Using PNZ and PNZ-Al3+, the concentrations of Al3+ and F- ions can be detected down to 2.89×10-7 M and 1.88×10-7 M, respectively. The cytotoxicity of the PNZ and its ability to bioimage Al3+ and F- ions was examined in the human cervical cancer cell line. Finally, the receptor PNZ was applied for the quantification of Al3+ and F- ions in various real samples, such as tap water, river water, rainwater, mouthwash, and toothpaste.

Exploring the Anticancer Potential of Phenolic nor-Triterpenes from Celastraceae Species.

To explore new compounds with antitumour activity, fifteen phenolic nor-tripterpenes isolated from Celastraceae species, Maytenus jelskii, Maytenus cuzcoina, and Celastrus vulcanicola, have been studied. Their chemical structures were elucidated through spectroscopic and spectrometric techniques, resulting in the identification of three novel chemical compounds. Evaluation on human tumour cell lines (A549 and SW1573, non-small cell lung; HBL-100 and T-47D, breast; HeLa, cervix, and WiDr, colon) revealed that three compounds, named 6-oxo-pristimerol, demethyl-zeylasteral, and zeylasteral, exhibited significant activity (GI50 ranging from 0.45 to 8.6 µM) on at least five of the cell lines tested. Continuous live cell imaging identified apoptosis as the mode of action of selective cell killing in HeLa cells. Furthermore, their effect on a drug-sensitive Saccharomyces cerevisiae strain has been investigated to deepen on their mechanism of action. In dose-response growth curves, zeylasteral and 7α-hydroxy-blepharodol were markedly active. Additionally, halo assays were conducted to assess the involvement of oxidative stress and/or mitochondrial function in the anticancer profile, ruling out these modes of action for the active compounds. Finally, we also delve into the structure-activity relationship, providing insights into how the molecular structure of these compounds influences their biological activity. This comprehensive analysis enhances our understanding of the therapeutic potential of this triterpene type and underscores its relevance for further research in this field.

Spatiotemporal dynamics of microscopic biological barrier visualized by electric-double-layer modulation imaging.

Live-cell label-free imaging of a microscopic biological barrier, generally referred to as 'tight junction', was realized by a recently developed electric-double-layer modulation imaging (EDLMI). The method allowed quantitative imaging of barrier integrity in real time, thus being an upper compatible of transepithelial electrical resistance (TEER) which is a conventional standard technique to evaluate spatially averaged barrier integrity. We demonstrate that the quantitative and real-time imaging capability of EDLMI unveils fundamental dynamics of biological barrier, some of which are totally different from conventional understandings.

CO2-Free On-Stage Incubator for Live Cell Imaging of Cholangiocarcinoma Cell Migration on Microfluidic Device.

Long-term live cell imaging requires sophisticated and fully automated commercial-stage incubators equipped with specified inverted microscopes to regulate temperature, CO2 content, and humidity. In this study, we present a CO2-free on-stage incubator specifically designed for use across various cell culture platforms, enabling live cell imaging applications. A simple and transparent incubator was fabricated from acrylic sheets to be easily placed on the stages of most inverted microscopes. We successfully performed live-cell imaging of cholangiocarcinoma (CCA) cells and HeLa cell dynamics in both 2D and 3D microenvironments over three days. We also analyzed directed cell migration under high serum induction within a microfluidic device. Interesting phenomena such as "whole-colony migration", "novel type of collective cell migration" and "colony formation during cell and colony migration" are reported here for the first time, to the best of our knowledge. These phenomena may improve our understanding of the nature of cell migration and cancer metastasis.

A Novel Method for Separating Full and Empty Adeno-Associated Viral Capsids Using Ultrafiltration.

Adeno-associated viral vectors (AAVs) are the predominant viral vectors used for gene therapy applications. A significant challenge in obtaining effective doses is removing non-therapeutic empty viral capsids lacking DNA cargo. Current methods for separating full (gene-containing) and empty capsids are challenging to scale, produce low product yields, are slow, and are difficult to operationalize for continuous biomanufacturing. This communication demonstrates the feasibility of separating full and empty capsids by ultrafiltration. Separation performance was quantified by measuring the sieving coefficients for full and empty capsids using ELISA, qPCR, and an infectivity assay based on the live cell imaging of green fluorescent protein expression. We demonstrated that polycarbonate track-etched membranes with a pore size of 30 nm selectively permeated empty capsids to full capsids, with a high recovery yield (89%) for full capsids. The average sieving coefficients of full and empty capsids obtained through ELISA/qPCR were calculated as 0.25 and 0.49, indicating that empty capsids were about twice as permeable as full capsids. Establishing ultrafiltration as a viable unit operation for separating full and empty AAV capsids has implications for developing the scale-free continuous purification of AAVs.

A comprehensive review on colorimetric and fluorometric investigations of dual sensing chemosensors for Cu2+ and Fe3+ ions from the year 2017 to 2023.

Dual sensing chemosensors for copper(II) and iron(III) ions are molecules or compounds designed to selectively detect and differentiate between these specific metal ions. Because metal ions like copper(II) and iron(III) are essential to so many industrial, biological, and environmental processes, their detection and measurement have become increasingly important. In this work, a novel dual-sensing chemosensor that combines high selectivity and sensitivity is presented. It is intended to detect copper(II) (Cu2+) and iron (III)(Fe3+) ions concurrently. The chemosensor combines two different recognition components into one platform and achieves dual-mode detection by combining optical and electrochemical sensing approaches. Using a dual sensing chemosensors for two cations can save money and time compared to preparing two separate chemosensors to sense each of those cations separately. We often use various techniques, including spectroscopy, fluorescence, and electrochemistry, to monitor and measure the changes induced by the interaction between the chemosensors and the metal ions. Discussions have been held on the excitation and emission wavelengths, media used in the spectroscopic measurements, binding constant with coordination binding mode, detection mechanism, and detection limit (LOD). This extensive review paper investigates colorimetric and fluorometric dual sensing analysis for Cu2+ and Fe3+ ions which includes more than sixty papers from the year of 2017 to 2023.

Expediting adenovirus titer assays via an algorithmic live-cell imaging technique.

Interest in virus-based therapeutics for the treatment of genetic and oncolytic diseases has created a demand for high-yield, low-cost virus-manufacturing processes. However, traditional analytical methods of assessing infectious virus titer require multiple processing steps and manual counting, limiting sample throughput, and increasing human error. This bottleneck severely limits the development of new manufacturing unit operations to drive down costs. In this work, we utilize an Incucyte Live-Cell Analysis System to develop a high-throughput infectious titer assay for adenovirus expressing a GFP-transgene. Although previous studies have demonstrated live-cell imaging's potential for use with other viruses, they provide little guidance regarding the selection of the viewing and analysis parameters. To fill this gap, we develop an algorithmic approach to identify the optimum viewing and analysis parameters and create a statistical workflow for quantifying infectious adenovirus in a sample dilution series in a standard 24-well microplate. The developed assay has a Pearson correlation coefficient of 0.9, which is comparable to Hexon staining, the gold-standard for adenovirus infectious titer. Finally, the developed algorithmic approach and statistical workflow were applied to create an assay for adenovirus titer using a 96-well microplate, allowing five times more samples to be quantified compared to the standard 24-well plate. While this assay uses a GFP-insert that precludes its use in a clinical environment, the key learnings surrounding the careful use of viewing and analysis parameters, and the statistical workflow are widely applicable to implementing life-cell imaging for dilution-series-based assays. Moreover, this method directly enables the fast and accurate evaluation of virus samples in a preclinical environment.

ECM stiffness regulates calcium influx into mitochondria via tubulin and VDAC1 activity.

Calcium ions (Ca2+) play pivotal roles in regulating numerous cellular functions, including metabolism and growth, in normal and cancerous cells. Consequently, Ca2+ signaling is a vital determinant of cell fate and influences both cell survival and death. These intracellular signals are susceptible to modulation by various factors, including changes in the extracellular environment, which leads to mechanical alterations. However, the effect of extracellular matrix (ECM) stiffness variations on intracellular Ca2+ signaling remains underexplored. In this study, we aimed to elucidate the mechanisms of Ca2+ regulation through the mitochondria, which are crucial to Ca2+ homeostasis. We investigated how Ca2+ regulatory mechanisms adapt to different levels of ECM stiffness by simultaneously imaging the mitochondria and endoplasmic reticulum (ER) in live cells using genetically encoded biosensors. Our findings revealed that the uptake of mitochondrial Ca2+ through Voltage-Dependent Anion Channel 1 (VDAC1), facilitated by intracellular tubulin, is influenced by ECM stiffness. Unraveling these Ca2+ regulatory mechanisms under various conditions offers a novel perspective for advancing biomedical studies involving Ca2+ signaling.

Super-resolution microscopy to study membrane nanodomains and transport mechanisms in the plasma membrane.

Biological membranes are complex, heterogeneous, and dynamic systems that play roles in the compartmentalization and protection of cells from the environment. It is still a challenge to elucidate kinetics and real-time transport routes for molecules through biological membranes in live cells. Currently, by developing and employing super-resolution microscopy; increasing evidence indicates channels and transporter nano-organization and dynamics within membranes play an important role in these regulatory mechanisms. Here we review recent advances and discuss the major advantages and disadvantages of using super-resolution microscopy to investigate protein organization and transport within plasma membranes.

Inhibitory effects of sucrose palmitic acid ester on the germination-to-outgrowth process of Clostridium perfringens SM101 spores.

As a commercially available esterified compound derived from sucrose and palmitoyl acids, sucrose ester palmitic acid (SEPA) has been used as an emulsifier in food processing. It possesses antibacterial activity against vegetative and spore-forming bacteria, including Clostridium, Moorella, Bacillus, and Geobacillus species, prompting the food industry to use it as a food additive to achieve a desirable shelf life; however, the precise mechanism by which the compound affects the physiological processes of bacteria and how it inhibits bacterial growth remains unclear. In this study, we focused on the inhibitory effect of SEPA on the germination-to-outgrowth process of Clostridium perfringens SM101 spores, a strain widely used as a model of C. perfringens. When the isolated spores were exposed to ≧ 20 µg/ml of SEPA on brain heart infusion agar, bacterial colony formation was completely inhibited. Time-resolved phase-contrast microscopy was employed to visualize the effect of SEPA on the entire regrowth process of SM101 spores. SEPA did not affect the "germination stage," where each spore changes its optical density from phase-bright to phase-dark. In contrast, the presence of SEPA completely blocked the "outgrowth stage," in which the newly synthesized vegetative cell body emerges from the cracked spore shell. The results demonstrate that SEPA inhibits the revival process of the spores of a pathogenic strain of C. perfringens and that the site of its action is the "outgrowth stage" and not the "germination stage," as evidenced by single- cell analysis.

HD-ZIP IV genes are essential for embryo initial cell polarization and the radial axis formation in Arabidopsis.

Plants develop along apical-basal and radial axes. In Arabidopsis thaliana, the radial axis becomes evident when the cells of the 8-cell proembryo divide periclinally, forming inner and outer cell layers. Although changes in cell polarity or morphology likely precede this oriented cell division, the initial events and the factors regulating radial axis formation remain elusive. Here, we report that three transcription factors belonging to the class IV homeodomain-leucine zipper (HD-ZIP IV) family redundantly regulate radial pattern formation: HOMEODOMAIN GLABROUS11 (HDG11), HDG12, and PROTODERMAL FACTOR2 (PDF2). The hdg11 hdg12 pdf2 triple mutant failed to undergo periclinal division at the 8-cell stage and cell differentiation along the radial axis. Live-cell imaging revealed that the mutant defect is already evident in the behavior of the embryo's initial cell (apical cell), which is generated by zygote division. In the wild type, the apical cell grows longitudinally and then radially, and its nucleus remains at the bottom of the cell, where the vertical cell plate emerges. By contrast, the mutant apical cell elongates longitudinally, and its nucleus releases from its basal position, resulting in a transverse division. Computer simulations based on the live-cell imaging data confirmed the importance of the geometric rule (the minimal plane principle and nucleus-passing principle) in determining the cell division plane. We propose that HDG11, HDG12, and PDF2 promote apical cell polarization, i.e., radial cell growth and basal nuclear retention, and set proper radial axis formation during embryogenesis.

ExoJ: an ImageJ2/Fiji plugin for automated spatiotemporal detection and analysis of exocytosis.

Exocytosis is a dynamic physiological process that enables the release of biomolecules to the surrounding environment via the fusion of membrane compartments to the plasma membrane. Understanding its mechanisms is crucial, as defects can compromise essential biological functions. The development of pH-sensitive optical reporters alongside fluorescence microscopy enables the assessment of individual vesicle exocytosis events at the cellular level. Manual annotation represents, however, a time-consuming task, prone to selection biases and human operational errors. Here, we introduce ExoJ, an automated plugin based on ImageJ2/Fiji. ExoJ identifies user-defined genuine populations of exocytosis events, recording quantitative features including intensity, apparent size and duration. We designed ExoJ to be fully user-configurable, making it suitable to study distinct forms of vesicle exocytosis regardless of the imaging quality. Our plugin demonstrates its capabilities by showcasing distinct exocytic dynamics among tetraspanins and vesicular SNAREs protein reporters. Assessment of performance on synthetic data showed ExoJ is a robust tool, capable to correctly identify exocytosis events independently of signal-to-noise ratio conditions. We propose ExoJ as a standard solution for future comparative and quantitative studies of exocytosis.

Interpretable Fine-Grained Phenotypes of Subcellular Dynamics via Unsupervised Deep Learning.

Uncovering fine-grained phenotypes of live cell dynamics is pivotal for a comprehensive understanding of the heterogeneity in healthy and diseased biological processes. However, this endeavor poses significant technical challenges for unsupervised machine learning, requiring the extraction of features that not only faithfully preserve this heterogeneity but also effectively discriminate between established biological states, all while remaining interpretable. To tackle these challenges, a self-training deep learning framework designed for fine-grained and interpretable phenotyping is presented. This framework incorporates an unsupervised teacher model with interpretable features to facilitate feature learning in a student deep neural network (DNN). Significantly, an autoencoder-based regularizer is designed to encourage the student DNN to maximize the heterogeneity associated with molecular perturbations. This method enables the acquisition of features with enhanced discriminatory power, while simultaneously preserving the heterogeneity associated with molecular perturbations. This study successfully delineated fine-grained phenotypes within the heterogeneous protrusion dynamics of migrating epithelial cells, revealing specific responses to pharmacological perturbations. Remarkably, this framework adeptly captured a concise set of highly interpretable features uniquely linked to these fine-grained phenotypes, each corresponding to specific temporal intervals crucial for their manifestation. This unique capability establishes it as a valuable tool for investigating diverse cellular dynamics and their heterogeneity.

Live-cell imaging and CLEM reveal the existence of ACTN4-dependent ruffle-edge lamellipodia acting as a novel mode of cell migration.

α-Actinin-4 (ACTN4) expression levels are correlated with the invasive and metastatic potential of cancer cells; however, the underlying mechanism remains unclear. Here, we identified ACTN4-localized ruffle-edge lamellipodia using live-cell imaging and correlative light and electron microscopy (CLEM). BSC-1 cells expressing EGFP-ACTN4 showed that ACTN4 was most abundant in the leading edges of lamellipodia, although it was also present in stress fibers and focal adhesions. ACTN4 localization in lamellipodia was markedly diminished by phosphoinositide 3-kinase inhibition, whereas its localization in stress fibers and focal adhesions remained. Furthermore, overexpression of ACTN4, but not ACTN1, promoted lamellipodial formation. Live-cell analysis demonstrated that ACTN4-enriched lamellipodia are highly dynamic and associated with cell migration. CLEM revealed that ACTN4-enriched lamellipodia exhibit a characteristic morphology of multilayered ruffle-edges that differs from canonical flat lamellipodia. Similar ruffle-edge lamellipodia were observed in A549 and MDA-MB-231 invasive cancer cells. ACTN4 knockdown suppressed the formation of ruffle-edge lamellipodia and cell migration during wound healing in A549 monolayer cultures. Additionally, membrane-type 1 matrix metalloproteinase was observed in the membrane ruffles, suggesting that ruffle-edge lamellipodia have the ability to degrade the extracellular matrix and may contribute to active cell migration/invasion in certain cancer cell types.

Next-generation 3D tumor modeling: A microfluidic platform with biocompatible red carbon dots for live cell imaging in co-cultured elongated spheroid tumor model.

Co-culture spheroids mimic tumor architecture more accurately than traditional 2D cell cultures, but non-invasive, long-term tracking of live cells within these 3D models remains a challenge. This study addresses this critical need by developing a novel approach for live cell imaging in U-87/HUF co-culture spheroids. We introduce water-soluble, biocompatible red carbon dots (R-CDs) exhibiting exceptional stability and brightness (21% quantum yield) specifically designed for imaging within these 3D models. Furthermore, we designed a microfluidic chip with ellipsoid-shaped microwells to efficiently generate two distinct co-culture spheroid types: direct mixing and core-shell. R-CDs enabled non-invasive tracking of U-87 cancer cell location within these 3D models demonstrating their efficacy for long-term monitoring of live cells in cancer research. This R-CD and microfluidic technology has the potential to accelerate cancer drug discovery by enabling live cell studies in 3D tumor models.

Cell fate simulation reveals cancer cell features in the tumor microenvironment.

To elucidate the dynamic evolution of cancer cell characteristics within the tumor microenvironment (TME), we developed an integrative approach combining single-cell tracking, cell fate simulation, and 3D TME modeling. We began our investigation by analyzing the spatiotemporal behavior of individual cancer cells in cultured pancreatic (MiaPaCa2) and cervical (HeLa) cancer cell lines, with a focus on the α2-6 sialic acid (α2-6Sia) modification on glycans, which is associated with cell stemness. Our findings revealed that MiaPaCa2 cells exhibited significantly higher levels of α2-6Sia modification, correlating with enhanced reproductive capabilities, whereas HeLa cells showed less prevalence of this modification. To accommodate the in vivo variability of α2-6Sia levels, we employed a cell fate simulation algorithm that digitally generates cell populations based on our observed data while varying the level of sialylation, thereby simulating cell growth patterns. Subsequently, we performed a 3D TME simulation with these deduced cell populations, considering the microenvironment that could impact cancer cell growth. Immune cell landscape information derived from 193 cervical and 172 pancreatic cancer cases was used to estimate the degree of the positive or negative impact. Our analysis suggests that the deduced cells generated based on the characteristics of MiaPaCa2 cells are less influenced by the immune cell landscape within the TME compared to those of HeLa cells, highlighting that the fate of cancer cells is shaped by both the surrounding immune landscape and the intrinsic characteristics of the cancer cells.

Diverse calcium signaling profiles regulate migratory behavior in avascular wound healing and aberrant signal hierarchy occurs early in diabetes.

In avascular wound repair, calcium signaling events are the predominant mechanism cells use to transduce information about stressors in the environment into an effective and coordinated migratory response. Live cell imaging and computational analysis of corneal epithelial wound healing revealed that signal initiation and propagation at the wound edge are highly ordered, with groups of cells engaging in cyclical patterns of initiation and propagation. The cells in these groups exhibit a diverse range of signaling behavior, and dominant "conductor cells" drive activity in groups of lower-signaling neighbors. Ex vivo model systems reveal that conductor cells are present in wing cell layers of the corneal epithelium and that signaling propagates both within and between wing and basal layers. There are significant aberrations in conductor phenotype and interlayer propagation in type II diabetic murine models, indicating that signal hierarchy breakdown is an early indicator of disease. In vitro models reveal that signaling profile diversity and conductor cell phenotype is eliminated with P2X7 inhibition and is altered in Pannexin-1 or P2Y2 but not Connexin-43 inhibition. Conductor cells express significantly less P2X7 than their lower-signaling neighbors and exhibit significantly less migratory behavior after injury. Together, our results show that the postinjury calcium signaling cascade exhibits significantly more ordered and hierarchical behavior than previously thought, that proteins previously shown to be essential for regulating motility are also essential for determining signaling phenotype, and that loss of signal hierarchy integrity is an early indicator of disease state. NEW & NOTEWORTHY Calcium signaling in corneal epithelial cells after injury is highly ordered, with groups of cells engaged in cyclical patterns of event initiation and propagation driven by high-signaling cells. Signaling behavior is determined by P2X7, Pannexin-1, and P2Y2 and influences migratory behavior. Signal hierarchy is observed in healthy ex vivo models after injury and becomes aberrant in diabetes. This represents a paradigm shift, as signaling was thought to be random and determined by factors in the environment.

Dynamic monitoring of viral gene expression reveals rapid antiviral effects of CD8 T cells recognizing the HCMV-pp65 antigen.

Introduction: Human Cytomegalovirus (HCMV) is a betaherpesvirus that causes severe disease in immunocompromised transplant recipients. Immunotherapy with CD8 T cells specific for HCMV antigens presented on HLA class-I molecules is explored as strategy for long-term relief to such patients, but the antiviral effectiveness of T cell preparations cannot be efficiently predicted by available methods. Methods: We developed an Assay for Rapid Measurement of Antiviral T-cell Activity (ARMATA) by real-time automated fluorescent microscopy and used it to study the ability of CD8 T cells to neutralize HCMV and control its spread. As a proof of principle, we used TCR-transgenic T cells specific for the immunodominant HLA-A02-restricted tegumental phosphoprotein pp65. pp65 expression follows an early/late kinetic, but it is not clear at which stage of the virus cycle it acts as an antigen. We measured control of HCMV infection by T cells as early as 6 hours post infection (hpi). Results: The timing of the antigen recognition indicated that it occurred before the late phase of the virus cycle, but also that virion-associated pp65 was not recognized during virus entry into cells. Monitoring of pp65 gene expression dynamics by reporter fluorescent genes revealed that pp65 was detectable as early as 6 hpi, and that a second and much larger bout of expression occurs in the late phase of the virus cycle by 48 hpi. Since transgenic (Tg)-pp65 specific CD8 T cells were activated even when DNA replication was blocked, our data argue that pp65 acts as an early virus gene for immunological purposes. Discussion: ARMATA does not only allow same day identification of antiviral T-cell activity, but also provides a method to define the timing of antigen recognition in the context of HCMV infection.