Super-Resolution Ultrasound Imaging Using the Erythrocytes-Part I: Density Images.

A new approach for vascular super-resolution (SR) imaging using the erythrocytes as targets (SUper-Resolution ultrasound imaging of Erythrocytes (SURE) imaging) is described and investigated. SURE imaging does not require fragile contrast agent bubbles, making it possible to use the maximum allowable mechanical index (MI) for ultrasound scanning for an increased penetration depth. A synthetic aperture (SA) ultrasound sequence was employed with 12 virtual sources (VSs) using a 10-MHz GE L8-18i-D linear array hockey stick probe. The axial resolution was [Formula: see text]m) and the lateral resolution was [Formula: see text]m). Field IIpro simulations were conducted on 12.5- µ m radius vessel pairs with varying separations. A vessel pair with a separation of 70 µ m could be resolved, indicating a SURE image resolution below half a wavelength. A Verasonics research scanner was used for the in vivo experiments to scan the kidneys of Sprague-Dawley rats for up to 46 s to visualize their microvasculature by processing from 0.1 up to 45 s of data for SURE imaging and for 46.8 s for SR imaging with a SonoVue contrast agent. Afterward, the renal vasculature was filled with the ex vivo micro-computed tomography (CT) contrast agent Microfil, excised, and scanned in a micro-CT scanner at both a 22.6- µ m voxel size for 11 h and for 20 h in a 5- µ m voxel size for validating the SURE images. Comparing the SURE and micro-CT images revealed that vessels with a diameter of 28 µ m, five times smaller than the ultrasound wavelength, could be detected, and the dense grid of microvessels in the full kidney was shown for scan times between 1 and 10 s. The vessel structure in the cortex was also similar to the SURE and SR images. Fourier ring correlation (FRC) indicated a resolution capability of 29 µ m. SURE images are acquired in seconds rather than minutes without any patient preparation or contrast injection, making the method translatable to clinical use.

Super-Resolution Ultrasound Imaging Using the Erythrocytes-Part II: Velocity Images.

Super-resolution ultrasound imaging using the erythrocytes (SURE) has recently been introduced. The method uses erythrocytes as targets instead of fragile microbubbles (MBs). The abundance of erythrocyte scatterers makes it possible to acquire SURE data in just a few seconds compared with several minutes in ultrasound localization microscopy (ULM) using MBs. A high number of scatterers can reduce the acquisition time; however, the tracking of uncorrelated and high-density scatterers is quite challenging. This article hypothesizes that it is possible to detect and track erythrocytes as targets to obtain vascular flow images. A SURE tracking pipeline is used with modules for beamforming, recursive synthetic aperture (SA) imaging, motion estimation, echo canceling, peak detection, and recursive nearest-neighbor (NN) tracker. The SURE tracking pipeline is capable of distinguishing the flow direction and separating tubes of a simulated Field II phantom with 125-25- [Formula: see text] wall-to-wall tube distances, as well as a 3-D printed hydrogel micr-flow phantom with 100-60- [Formula: see text] wall-to-wall channel distances. The comparison of an in vivo SURE scan of a Sprague-Dawley rat kidney with ULM and micro-computed tomography (CT) scans with voxel sizes of 26.5 and [Formula: see text] demonstrated consistent findings. A microvascular structure composed of 16 vessels exhibited similarities across all imaging modalities. The flow direction and velocity profiles in the SURE scan were found to be concordant with those from ULM.

Generation of three isogenic gene-edited Huntington's disease human embryonic stem cell lines with DOX-inducible NGN2 expression cassette in the AAVS1 safe locus.

Neurogenin 2 (NGN2), a neuronal transcription factor, can expedite differentiation of stem cells into mature glutamatergic neurons. We have utilized an allelic series of previously published and characterized isogenic Huntington's disease (IsoHD) human embryonic stem cell lines (Ooi et al., 2019), carrying different CAG repeat lengths in the first exon of the huntingtin gene. These IsoHDs were modified using CRISPR/Cas9 to insert NGN2 under the TET-ON doxycycline inducible promoter. The resulting IsoHD-NGN2 cell lines retained pluripotency in the absence of doxycycline (DOX), and via addition of DOX to the culturing media differentiation to neurons was achieved within 14 days.

Super-Resolution Ultrasound Imaging of Renal Vascular Alterations in Zucker Diabetic Fatty Rats during the Development of Diabetic Kidney Disease.

Individuals with diabetes at risk of developing diabetic kidney disease (DKD) are challenging to identify using currently available clinical methods. Prognostic accuracy and initiation of treatment could be improved by a quantification of the renal microvascular rarefaction and the increased vascular tortuosity during the development of DKD. Super-resolution ultrasound (SRUS) imaging is an in vivo technique capable of visualizing blood vessels at sizes below 75 µm. This preclinical study aimed to investigate the alterations in renal blood vessels' density and tortuosity in a type 2 diabetes rat model, Zucker diabetic fatty (ZDF) rats, as a prediction of DKD. Lean age-matched Zucker rats were used as controls. A total of 36 rats were studied, subdivided into ages of 12, 22, and 40 weeks. Measured albuminuria indicated the early stage of DKD, and the SRUS was compared with the ex vivo micro-computed tomography (µCT) of the same kidneys. Assessed using the SRUS imaging, a significantly decreased cortical vascular density was detected in the ZDF rats from 22 weeks of age compared to the healthy controls, concomitant with a significantly increased albuminuria. Already by week 12, a trend towards a decreased cortical vascular density was found prior to the increased albuminuria. The quantified vascular density in µCT corresponded with the in vivo SRUS imaging, presenting a consistently lower vascular density in the ZDF rats. Regarding vessel tortuosity, an overall trend towards an increased tortuosity was present in the ZDF rats. SRUS shows promise for becoming an additional tool for monitoring and prognosing DKD. In the future, large-scale animal studies and human trials are needed for confirmation.

Engineered lentivirus-derived nanoparticles (LVNPs) for delivery of CRISPR/Cas ribonucleoprotein complexes supporting base editing, prime editing and in vivo gene modification.

Implementation of therapeutic in vivo gene editing using CRISPR/Cas relies on potent delivery of gene editing tools. Administration of ribonucleoprotein (RNP) complexes consisting of Cas protein and single guide RNA (sgRNA) offers short-lived editing activity and safety advantages over conventional viral and non-viral gene and RNA delivery approaches. By engineering lentivirus-derived nanoparticles (LVNPs) to facilitate RNP delivery, we demonstrate effective administration of SpCas9 as well as SpCas9-derived base and prime editors (BE/PE) leading to gene editing in recipient cells. Unique Gag/GagPol protein fusion strategies facilitate RNP packaging in LVNPs, and refinement of LVNP stoichiometry supports optimized LVNP yield and incorporation of therapeutic payload. We demonstrate near instantaneous target DNA cleavage and complete RNP turnover within 4 days. As a result, LVNPs provide high on-target DNA cleavage and lower levels of off-target cleavage activity compared to standard RNP nucleofection in cultured cells. LVNPs accommodate BE/sgRNA and PE/epegRNA RNPs leading to base editing with reduced bystander editing and prime editing without detectable indel formation. Notably, in the mouse eye, we provide the first proof-of-concept for LVNP-directed in vivo gene disruption. Our findings establish LVNPs as promising vehicles for delivery of RNPs facilitating donor-free base and prime editing without formation of double-stranded DNA breaks.

Comparison of prevalence and mental health problems across symptom frequency of self-reported symptoms of binge-eating disorder in a community sample of adolescents.

OBJECTIVE: Binge-eating disorder (BED) and subthreshold BED (SBED) are prevalent in adults and associated with mental health problems including depression, non-suicidal self-injury, lower quality of life, and suicidality. There is solid evidence that binge-eating behaviors are also prevalent in adolescence, but knowledge about mental health in community adolescents with BED of different frequency thresholds is more limited. We aimed to investigate the prevalence and mental health problems associated with SBED of low frequency and/or limited duration compared with BED in a Danish community sample of adolescents. METHODS: We included 2509 adolescents who completed the online survey of the 16-17-year follow-up of the Copenhagen Child Cohort (CCC2000), including items on BED symptoms approximating the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) criteria, and items on mental health and quality of life. RESULTS: The 1-year prevalence of SBED was 2.7% (95% confidence interval [CI]: 2.0%-3.3%) with a male:female ratio of 1:3.7; comparable to previous findings on BED in the same sample. SBED was also comparable to BED concerning cross-sectional associations with overall mental health problems, lower health-related quality of life, depressive symptoms, and suicidal ideation, whereas no associations were seen with non-suicidal self-injury after Holm-Bonferroni correction. In both groups, thoughts and behaviors concerning food and weight interfered significantly with daily life. DISCUSSION: SBED and BED were equally prevalent in this adolescent community sample, and similarly associated with indicators of poor mental health. The findings indicate that community adolescents reporting symptoms approximating clinical criteria of BED need intervention irrespectively of symptom frequency or duration. PUBLIC SIGNIFICANCE: This study adds knowledge to the field by comparing BED of low frequency and/or limited duration ("subthreshold BED," SBED) with full-syndrome BED in adolescents and showing that SBED in adolescence is both prevalent and associated with poor mental health to a similar extent as that of BED. Findings indicate that self-reported symptoms according to clinical criteria of SBED and BED alike constitute a public health problem and point to youngsters in need of intervention.

Aagenaes syndrome/lymphedema cholestasis syndrome 1 is caused by a founder variant in the 5'-untranslated region of UNC45A.

BACKGROUND & AIMS: Lymphedema cholestasis syndrome 1 or Aagenaes syndrome is a condition characterized by neonatal cholestasis, lymphedema, and giant cell hepatitis. The genetic background of this autosomal recessive disease was unknown up to now. METHODS: A total of 26 patients with Aagenaes syndrome and 17 parents were investigated with whole-genome sequencing and/or Sanger sequencing. PCR and western blot analyses were used to assess levels of mRNA and protein, respectively. CRISPR/Cas9 was used to generate the variant in HEK293T cells. Light microscopy, transmission electron microscopy and immunohistochemistry for biliary transport proteins were performed in liver biopsies. RESULTS: One specific variant (c.-98G>T) in the 5'-untranslated region of Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome. Nineteen were homozygous for the c.-98G>T variant and seven were compound heterozygous for the variant in the 5'-untranslated region and an exonic loss-of-function variant in UNC45A. Patients with Aagenaes syndrome exhibited lower expression of UNC45A mRNA and protein than controls, and this was reproduced in a CRISPR/Cas9-created cell model. Liver biopsies from the neonatal period demonstrated cholestasis, paucity of bile ducts and pronounced formation of multinucleated giant cells. Immunohistochemistry revealed mislocalization of the hepatobiliary transport proteins BSEP (bile salt export pump) and MRP2 (multidrug resistance-associated protein 2). CONCLUSIONS: c.-98G>T in the 5'-untranslated region of UNC45A is the causative genetic variant in Aagenaes syndrome. IMPACT AND IMPLICATIONS: The genetic background of Aagenaes syndrome, a disease presenting with cholestasis and lymphedema in childhood, was unknown until now. A variant in the 5'-untranslated region of the Unc-45 myosin chaperone A (UNC45A) was identified in all tested patients with Aagenaes syndrome, providing evidence of the genetic background of the disease. Identification of the genetic background provides a tool for diagnosis of patients with Aagenaes syndrome before lymphedema is evident.

A 1-year follow-up study in patients with idiopathic pulmonary fibrosis regarding adverse outcomes to unintended weight loss.

OBJECTIVES: Malnutrition in pulmonary fibrosis may influence clinical outcomes negatively. This project aimed to investigate if weight, unintended weight loss (UWL) at baseline and weight development, and signs of sarcopenia measured by the strength, assistance with the walking, rising from a chair, climbing stairs, and falls questionnaire (SARC-F) are associated with hospital admissions and mortality for idiopathic pulmonary fibrosis outpatients in ≤1 y as well as referral to pulmonary rehabilitation. METHODS: At baseline, prevalence of weight and UWL were sought in a cross-sectional questionnaire study, consecutively, including 100 patients in an outpatient clinic. Medical records were sought for time from diagnosis and comorbidities. One year after inclusion weight, UWL and SARC-F were collected by phone interviews, and medical records were revisited for clinical outcomes. RESULTS: Of the 100 patients, two patients died and seven were lost to follow-up. The prevalence of UWL increased within the year (10-13%), and the amount of UWL increased (9.1-11.8 kg). Patients with a UWL at baseline had a significantly higher risk of mortality (odds ratio = 29.8; P = 0.037). UWL at baseline was associated with risk of hospital admissions (odds ratio = 14.7; P = 0.009). Based on the results from SARC-F, 20.9% have signs of sarcopenia. UWL at follow-up was associated with the risk of sarcopenia by SARC-F. Patients with risk of sarcopenia and those with body mass index ≥30 kg/m2 were to a higher degree offered pulmonary rehabilitation; however, participation was low. CONCLUSIONS: UWL at baseline was significantly associated with risk of hospital admissions and mortality in ≤1 y in idiopathic pulmonary fibrosis outpatients. Patients with signs of sarcopenia and body mass index ≥30 kg/m2 were most often referred to pulmonary rehabilitation.

CRISPR-Cas9-directed gene tagging using a single integrase-defective lentiviral vector carrying a transposase-based Cas9 off switch.

Locus-directed DNA cleavage induced by the CRISPR-Cas9 system triggers DNA repair mechanisms allowing gene repair or targeted insertion of foreign DNA. For gene insertion to be successful, availability of a homologous donor template needs to be timed with cleavage of the DNA by the Cas9 endonuclease guided by a target-specific single guide RNA (sgRNA). We present a novel approach for targeted gene insertion based on a single integrase-defective lentiviral vector (IDLV) carrying a Cas9 off switch. Gene insertion using this approach benefits from transposon-based stable Cas9 expression, which is switched off by excision-only transposase protein co-delivered in IDLV particles carrying a combined sgRNA/donor vector. This one-vector approach supports potent (up to >80%) knockin of a full-length EGFP gene sequence. This traceless cell engineering method benefits from high stable levels of Cas9, timed intracellular availability of the molecular tools, and a built-in feature to turn off Cas9 expression after DNA cleavage. The simple technique is based on transduction with a single IDLV, which holds the capacity to transfer larger donor templates, allowing robust gene knockin or tagging of genes in a single step.

Super-Resolution Ultrasound Imaging Provides Quantification of the Renal Cortical and Medullary Vasculature in Obese Zucker Rats: A Pilot Study.

Obesity is a risk factor of chronic kidney disease (CKD), leading to alterations in the renal vascular structure. This study tested if renal vascular density and tortuosity was quantifiable in vivo in obese rats using microbubble-based super-resolution ultrasound imaging. The kidneys of two 11-week-old and two 20-week-old male obese Zucker rats were compared with age-matched male lean Zucker rats. The super-resolution ultrasound images were manually divided into inner medulla, outer medulla, and cortex, and each area was subdivided into arteries and veins. We quantified vascular density and tortuosity, number of detected microbubbles, and generated tracks. For comparison, we assessed glomerular filtration rate, albumin/creatinine ratio, and renal histology to evaluate CKD. The number of detected microbubbles and generated tracks varied between animals and significantly affected quantification of vessel density. In areas with a comparable number of tracks, density increased in the obese animals, concomitant with a decrease in glomerular filtration rate and an increase in albumin/creatinine ratio, but without any pathology in the histological staining. The results indicate that super-resolution ultrasound imaging can be used to quantify structural alterations in the renal vasculature. Techniques to generate more comparable number of microbubble tracks and confirmation of the findings in larger-scale studies are needed.

Super-Resolution Ultrasound Imaging Can Quantify Alterations in Microbubble Velocities in the Renal Vasculature of Rats.

Super-resolution ultrasound imaging, based on the localization and tracking of single intravascular microbubbles, makes it possible to map vessels below 100 µm. Microbubble velocities can be estimated as a surrogate for blood velocity, but their clinical potential is unclear. We investigated if a decrease in microbubble velocity in the arterial and venous beds of the renal cortex, outer medulla, and inner medulla was detectable after intravenous administration of the α1-adrenoceptor antagonist prazosin. The left kidneys of seven rats were scanned with super-resolution ultrasound for 10 min before, during, and after prazosin administration using a bk5000 ultrasound scanner and hockey-stick probe. The super-resolution images were manually segmented, separating cortex, outer medulla, and inner medulla. Microbubble tracks from arteries/arterioles were separated from vein/venule tracks using the arterial blood flow direction. The mean microbubble velocities from each scan were compared. This showed a significant prazosin-induced velocity decrease only in the cortical arteries/arterioles (from 1.59 ± 0.38 to 1.14 ± 0.31 to 1.18 ± 0.33 mm/s, p = 0.013) and outer medulla descending vasa recta (from 0.70 ± 0.05 to 0.66 ± 0.04 to 0.69 ± 0.06 mm/s, p = 0.026). Conclusively, super-resolution ultrasound imaging makes it possible to detect and differentiate microbubble velocity responses to prazosin simultaneously in the renal cortical and medullary vascular beds.

Ultrasound super-resolution imaging with a hierarchical Kalman tracker.

Microbubble (MB) tracking plays an important role in ultrasound super-resolution imaging (SRI) by enabling velocity estimation and improving image quality. This work presents a new hierarchical Kalman (HK) tracker to achieve better performance at scenarios with high concentrations of MBs and high localization uncertainty. The method attempts to follow MBs with different velocity ranges using different Kalman filters. An extended simulation framework for evaluating trackers is also presented and used for comparison of the proposed HK tracker with the nearest-neighbor (NN) and Kalman (K) trackers. The HK tracks were most similar to the ground truth with the highest Jaccard similarity coefficient in 79% of the scenarios and the lowest root-mean-square error in 72% of the scenarios. The HK tracker reconstructed vessels with a more accurate diameter. In a scenario with an uncertainty of 51.2µm in MB localization, a vessel diameter of 250µm was estimated as 257µm by HK tracker, compared with 329µm and 389µm for the K and NN trackers. In the same scenario, the HK tracker estimated MB velocities with a relative bias down to 1.7% and a relative standard deviation down to 8.3%. Finally, the different tracking techniques were applied to in vivo data from rat kidneys, and trends similar to the simulations were observed. Conclusively, the results showed an improvement in tracking performance, when the HK tracker was employed in comparison with the NN and K trackers.

Haematoma in the airways as the first symptom of acquired haemophilia A.

Acquired haemophilia A (AHA) is a rare autoimmune disorder resulting from antibodies against coagulation factor VIII. AHA causes severe, unexpected bleeding which may be life-threatening and should be considered when a patient with no previous history of bleeding presents with spontaneous bleeding and a prolonged APTT. This case report describes onset of AHA of a 75-year-old male who was admitted to Vejle Hospital with acute need for intubation due to breathing and swallowing problems caused by supraglottic haematoma. The case was complicated by anticoagulant treatment with rivaroxaban.

Evaluation of 2D super-resolution ultrasound imaging of the rat renal vasculature using ex vivo micro-computed tomography.

Super-resolution ultrasound imaging (SRUS) enables in vivo microvascular imaging of deeper-lying tissues and organs, such as the kidneys or liver. The technique allows new insights into microvascular anatomy and physiology and the development of disease-related microvascular abnormalities. However, the microvascular anatomy is intricate and challenging to depict with the currently available imaging techniques, and validation of the microvascular structures of deeper-lying organs obtained with SRUS remains difficult. Our study aimed to directly compare the vascular anatomy in two in vivo 2D SRUS images of a Sprague-Dawley rat kidney with ex vivo µCT of the same kidney. Co-registering the SRUS images to the µCT volume revealed visually very similar vascular features of vessels ranging from ~ 100 to 1300 µm in diameter and illustrated a high level of vessel branching complexity captured in the 2D SRUS images. Additionally, it was shown that it is difficult to use µCT data of a whole rat kidney specimen to validate the super-resolution capability of our ultrasound scans, i.e., validating the actual microvasculature of the rat kidney. Lastly, by comparing the two imaging modalities, fundamental challenges for 2D SRUS were demonstrated, including the complexity of projecting a 3D vessel network into 2D. These challenges should be considered when interpreting clinical or preclinical SRUS data in future studies.

piggyPrime: High-Efficacy Prime Editing in Human Cells Using piggyBac-Based DNA Transposition.

Prime editing is a novel genome editing technology that allows a wide range of tailored genomic alterations. Prime editing does not involve homologous recombination, but suffers from low efficacy. Here, we demonstrate piggyPrime, a transfected single-vector system based on piggyBac DNA transposition for genomic integration of all prime editing components in human cells allowing easy and effective transgenesis with prime editing efficacies up to 100% in cell lines.

Why Do Women with Eating Disorders Decline Treatment? A Qualitative Study of Barriers to Specialized Eating Disorder Treatment.

Despite the fact that eating disorders (EDs) are conditions that are potentially life-threatening, many people decline treatment. The aim of this study was to investigate why women decline specialized ED treatment, including their viewpoints on treatment services. Eighteen semi-structured qualitative interviews were conducted with women who had declined inpatient or outpatient specialized ED treatment. A thematic analysis revealed five main themes: (1) Disagreement on treatment needs, (2) rigid standard procedures, (3) failure to listen, (4) deprivation of identity, and (5) mistrust and fear. The women had declined ED treatment because they believed that treatment was only focused on nutritional rehabilitation and that it failed to address their self-identified needs. From their perspectives treatment was characterized by rigid standard procedures that could not be adapted to their individual situations and preferences. They felt that the therapists failed to listen to them, and they felt deprived of identity and reduced to an ED instead of a real person. This investigation is one of the first of its kind to provide clues as to how treatment could be moderated to better meet the needs of women who decline specialized ED treatment.

Highlights of the development in ultrasound during the last 70 years: A historical review.

This review looks at highlights of the development in ultrasound, ranging from interventional ultrasound and Doppler to the newest techniques like contrast-enhanced ultrasound and elastography, and gives reference to some of the valuable articles in Acta Radiologica. Ultrasound equipment is now available in any size and for any purpose, ranging from handheld devices to high-end devices, and the scientific societies include ultrasound professionals of all disciplines publishing guidelines and recommendations. Interventional ultrasound is expanding the field of use of ultrasound-guided interventions into nearly all specialties of medicine, from ultrasound guidance in minimally invasive robotic procedures to simple ultrasound-guided punctures performed by general practitioners. Each medical specialty is urged to define minimum requirements for equipment, education, training, and maintenance of skills, also for medical students. The clinical application of contrast-enhanced ultrasound and elastography is a topic often seen in current research settings.

In Vivo Motion Correction in Super-Resolution Imaging of Rat Kidneys.

Super-resolution (SR) imaging has the potential of visualizing the microvasculature down to the 10- [Formula: see text] level, but motion induced by breathing, heartbeats, and muscle contractions are often significantly above this level. This article, therefore, introduces a method for estimating tissue motion and compensating for this. The processing pipeline is described and validated using Field II simulations of an artificial kidney. In vivo measurements were conducted using a modified bk5000 research scanner (BK Medical, Herlev, Denmark) with a BK 9009 linear array probe employing a pulse amplitude modulation scheme. The left kidney of ten Sprague-Dawley rats was scanned during open laparotomy. A 1:10 diluted SonoVue contrast agent (Bracco, Milan, Italy) was injected through a jugular vein catheter at 100 [Formula: see text]/min. Motion was estimated using speckle tracking and decomposed into contributions from the heartbeats, breathing, and residual motion. The estimated peak motions and their precisions were: heart: axial- [Formula: see text] and lateral- [Formula: see text], breathing: axial- [Formula: see text] and lateral- [Formula: see text], and residual: axial-30 [Formula: see text] and lateral-90 [Formula: see text]. The motion corrected microbubble tracks yielded SR images of both bubble density and blood vector velocity. The estimation was, thus, sufficiently precise to correct shifts down to the 10- [Formula: see text] capillary level. Similar results were found in the other kidney measurements with a restoration of resolution for the small vessels demonstrating that motion correction in 2-D can enhance SR imaging quality.

Single-Cell Monitoring of Activated Innate Immune Signaling by a d2eGFP-Based Reporter Mimicking Time-Restricted Activation of IFNB1 Expression.

The innate immune system represents a balanced first line of defense against infection. Type I interferons (IFNs) are key regulators of the response to viral infections with an essential early wave of IFN-ß expression, which is conditional, time-restricted, and stochastic in its nature. The possibility to precisely monitor individual cells with active IFNB1 transcription during innate signaling requires a robust reporter system that mimics the endogenous IFN-ß signal. Here, we present a reporter system based on expression of a destabilized version of eGFP (d2eGFP) from a stably integrated reporter cassette containing the IFNB1 promoter and 3'-untranslated region, enabling both spatial and temporal detection of regulated IFNB1 expression. Specifically, this reporter permits detection, quantification, and isolation of cells actively producing d2eGFP in a manner that fully mimics IFN-ß production allowing tracking of IFNB1 gene activation and repression in monocytic cells and keratinocytes. Using induced d2eGFP expression as a readout for activated immune signaling at the single-cell level, we demonstrate the application of the reporter for FACS-based selection of cells with genotypes supporting cGAS-STING signaling. Our studies provide a novel approach for monitoring on/off-switching of innate immune signaling and form the basis for investigating genotypes affecting immune regulation at the single-cell level.