Bilirubin Distribution in Plants at the Subcellular and Tissue Levels.

In heterotrophs, heme degradation produces bilirubin, a tetrapyrrole compound that has antioxidant activity. In plants, heme is degraded in plastids and is believed to be converted to phytochromobilin rather than bilirubin. Recently, we used the bilirubin-inducible fluorescent protein UnaG to reveal that plants produce bilirubin via a non-enzymatic reaction with NADPH. In the present study, we used an UnaG-based live imaging system to visualize bilirubin accumulation in Arabidopsis thaliana and Nicotiana benthamiana at the organelle and tissue levels. In chloroplasts, bilirubin preferentially accumulated in the stroma, and the stromal bilirubin level increased upon dark treatment. Investigation of intracellular bilirubin distribution in leaves and roots showed that it accumulated mostly in plastids, with low levels detected in the cytosol and other organelles, such as peroxisomes, mitochondria and the endoplasmic reticulum. A treatment that increased bilirubin production in chloroplasts decreased the bilirubin level in peroxisomes, implying that a bilirubin precursor is transported between the two organelles. At the cell and tissue levels, bilirubin showed substantial accumulation in the root elongation region but little or none in the root cap and guard cells. Intermediate bilirubin accumulation was observed in other shoot and root tissues, with lower levels in shoot tissues. Our data revealed the distribution of bilirubin in plants, which has implications for the transport and physiological function of tetrapyrroles.

A guide to genetically-encoded redox biosensors: State of the art and opportunities.

Genetically-encoded redox biosensors have become invaluable tools for monitoring cellular redox processes with high spatiotemporal resolution, coupling the presence of the redox-active analyte with a change in fluorescence signal that can be easily recorded. This review summarizes the available fluorescence recording methods and presents an in-depth classification of the redox biosensors, organized by the analytes they respond to. In addition to the fluorescent protein-based architectures, this review also describes the recent advances on fluorescent, chemigenetic-based redox biosensors and other emerging chemigenetic strategies. This review examines how these biosensors are designed, the biosensors sensing mechanism, and their practical advantages and disadvantages.

Generation of a recombinant Saffold Virus expressing UnaG as a marker for the visualization of viral infection.

BACKGROUND: Saffold virus (SAFV), which belongs to the genus Cardiovirus of the family Picornaviridae, is associated with acute respiratory or gastrointestinal illnesses in children; it is also suspected to cause severe diseases, such as acute flaccid paralysis and aseptic meningitis. However, the understanding of the mechanism of its pathogenicity is still limited due to the many unknowns about its lifecycle; for example, the cellular receptor for its infection remains to be determined. A system to monitor SAFV infection in vitro and in vivo is required in order to accelerate research on SAFV. RESULTS: We generated a recombinant SAFV expressing green fluorescent protein (GFP) or UnaG, a novel fluorescent protein derived from Japanese eel. HeLa cells infected by either GFP or UnaG-expressing SAFV showed a bright green fluorescent signal, enabling convenient monitoring of SAFV infection. However, the expression of GFP but not UnaG was quickly lost during virus passaging due to the difference in genetic stability in the SAFV virus genome; the UnaG gene was stably maintained in the virus genome after at least five passages. CONCLUSIONS: SAFV infection of cultured cells can easily be monitored using UnaG-expressing SAFV, which is superior to GFP in terms of genetic stability in the virus genome. This virus could be a useful tool for SAFV research, such as comparing the susceptibility of various cells to SAFV infection and evaluating the effects of antivirals on SAFV infection in high-throughput screening.

The role of urinary N-acetyl-ß-D-glucosaminidase in early detection of acute kidney injury among pediatric patients with neoplastic disorders in a retrospective study.

BACKGROUND: The 1-year cumulative incidence of AKI reportedly is high (52%) in pediatric neoplastic disorders. About half of these events occur within 2 weeks. However, subclinical AKI episodes may remain unrecognized by the conventional creatinine-based approaches. We investigated the diagnostic value of urinary N-acetyl-ß-D-glucosaminidase (uNAG) as an early marker of acute kidney injury (AKI). METHODS: In our retrospective study, 33 children with neoplastic disorders were inculded who had serial uNAG tests (at least 5 samples/patient) with a total of 367 uNAG measurements. Renal function was determined by cystatin-C and creatinine based GFR, and relative increase of uNAG index (uNAGRI). We focused on detecting both clinical and subclinical AKI episodes (according to Biomarker-Guided Risk Assessment using pRIFLE criteria and /or elevated uNAG levels) and the incidence of chronic kidney damage. RESULTS: Sixty episodes in 26 patients, with positivity at least in one parameter of kidney panel, were identified during the observation period. We detected 18/60 clinical and 12/60 subclinical renal episodes. In 27/60 episodes only uNAG values was elevated with no therapeutic consequence at presentation. Two patients were detected with decreased initial creatinine levels with 3 "silent" AKI. In 13 patients, modest elevation of uNAG persisted suggesting mild, reversible tubular damage, while chronic tubuloglomerular injury occurred in 5 patients. Based on ROC analysis for the occurence of AKI, uNAGRI significantly indicated the presence of AKI, the sensitivity and specificity are higher than the changes of GFRCreat. Serial uNAG measurements are recommended for  the reduction of the great amount of false positive uNAG results, often due to overhydratation. CONCLUSION: Use of Biomarker-guided Risk Assessment for AKI identified 1.5 × more clinical and subclinical AKI episodes than with creatinine alone in our pediatric cancer patients. Based on the ROC curve for the occurence of AKI, uNAGRI has relatively high sensitivity and specificity comparable to changes of GFRCysC. The advantage of serial uNAG measurements is to decrease the number of false positive results. TRIAL REGISTRATION: The consent to participate is not applicable because it was not reqired for ethical approval and it is a retrospectiv study.

Investigation of mutations (L41F, F17M, N57E, Y99F_Y134W) effects on the TolAIII-UnaG fluorescence protein's unconjugated bilirubin (UC-BR) binding ability and thermal stability properties.

The UnaG protein is a ligand (unconjugated bilirubin) dependent fluorescence protein isolated from Unagi freshwater eel larvae and expressed as fusion in heterologous expression systems. Bilirubin is a tetrapyrrole molecule mainly produced from heme catabolism by the destruction of erythrocytes in the body. Bilirubin can cause kernicterus, a serious condition associated with permanent neurological damage in neonates with the passage of brain tissue. Different methods have been developed for plasma bilirubin analysis and quantification. The use of UnaG fluorescence protein triggered by bilirubin has become a new approach in bilirubin studies. In this study, we aimed to investigate the biophysical characterization of ligand interactions with the proteins obtained as a result of mutations (UnaGY99F_Y134W, UnaGN57E, UnaGL41F, and UnaGF17M) on the amino acid sequence of TolAIII-UnaG protein. After the purity levels of the expressed proteins have been analyzed by SDS-PAGE, secondary structures and thermal melting temperatures of the proteins have been examined by circular dichroism spectroscopy. Then determination of excitation and emission points by fluorescence spectroscopy, titration studies have been performed with bilirubin, and dissociation constant was calculated. According to the biophysical characterization studies, UnaGL41F has the highest affinity and stability among the mutants.

Molecular dynamics simulation of Toxin-Antitoxin (TA) system in Acinetobacter baumannii to explore the novel mechanism for inhibition of cell wall biosynthesis: Zeta Toxin as an effective therapeutic target.

The majority of bacteria and archaea contains Toxin-Antitoxin system (TA) that codes for the stable Toxin and unstable Antitoxin components forming a complex. The Antitoxin inhibits the catalytic activities of the Toxin. In general, the Antitoxin will be degraded by the proteases leading to the Toxin activation that subsequently targets essential cellular processes, including transcription, translation, replication, cell division, and cell wall biosynthesis. The Zeta Toxin-Epsilon Antitoxin system in ESKAPE pathogen stabilizes the resistance plasmid and promotes pathogenicity. The known TA system in Acinetobacter baumannii are known to be involved in the replication and translation, however, the mechanism of Zeta Toxin-Epsilon Antitoxin in cell wall biosynthesis remains unknown. In the present study, molecular docking and molecular dynamic (MD) simulations were employed to demonstrate whether Zeta Toxin can impair cell wall synthesis in A. baumannii. Further, the degradation mechanism of Antitoxin in the presence and absence of adenosine triphosphate (ATP) molecules are explained through MD simulation. The result reveals that the cleavage of Antitoxin could be possible with the presence of ATP by displaying its response from 20 ns, whereas the Zeta Toxin/Epsilon was unstable after 90 ns. The obtained results demonstrate that Zeta Toxin is "temporarily favorable" for ATP to undergo phosphorylation at UNAG kinase through the substrate tunneling process. The study further evidenced that phosphorylated UNAG prevents the binding of MurA, the enzyme that catalyzes the initial step of bacterial peptidoglycan biosynthesis. Therefore, the present study explores the binding mechanism of Zeta Toxin/Epsilon Antitoxin, which could be beneficial for preventing cell wall biosynthesis as well as for unveiling the alternative treatment options to antibiotics.

Renal function in ß-thalassemia major patients treated with two different iron-chelation regimes.

BACKGROUND: Renal injury in transfusion dependent ß thalassemia patients (TDT) has been attributed to iron overload, chronic anemia and iron-chelation therapy (ICT) toxicity. We studied renal function in TDT patients treated with two different ICT regimes. PATIENTS AND METHODS: We studied 36 TDT patients: 26 received deferasirox (DFX) and 10 were treated with deferoxamine (DFO) +/- deferiprone (DFP). RESULTS: Increased uNAG was found in 30% of the DFX group vs. 10% of the DFO+/-DFP group, the mean uNAG level in the DFX group was significantly higher than in the DFO+/-DFP group, (P < 0.05). A moderate negative correlation was found between uNAG levels and mean serum ferritin for the prior 10 years (P = 0.03), more pronounced for the DFO+/-DFP group. Twenty nine patients had had their renal function evaluated 10 years earlier; eGFR significantly declined in patients switched to DFX (P = 0.0093) but not in patients who continued DFO+/-DFP. CONCLUSIONS: A high prevalence of renal tubular damage was observed in our TDT patients, particularly those treated with DFX; uNAG was negatively associated with mean 10-year serum ferritin, suggesting ICT's involvement in tubular injury. A significant decline in eGFR compared to a decade earlier was observed only in patients currently treated with DFX. Strict follow-up of renal function in TDT patients is warranted.

A novel family of fluorescent hypoxia sensors reveal strong heterogeneity in tumor hypoxia at the cellular level.

Hypoxia is an intensively investigated condition with profound effects on cell metabolism, migration, and angiogenesis during development and disease. Physiologically, hypoxia is linked to tissue homeostasis and maintenance of pluripotency. Hypoxia also contributes to pathologies including cardiovascular diseases and cancer. Despite its importance, microscopic visualization of hypoxia is largely restricted to the detection of reductively activated probes by immunostaining. Here, we describe a novel family of genetically encoded fluorescent sensors that detect the activation of HIF transcription factors reported by the oxygen-independent fluorescent protein UnaG. It comprises sensors with different switching and memory behavior and combination sensors that allow the distinction of hypoxic and reoxygenated cells. We tested these sensors on orthotopically transplanted glioma cell lines. Using a cranial window, we could visualize hypoxia intravitally at cellular resolution. In tissue samples, sensor activity was detected in regions, which were largely devoid of blood vessels, correlated with HIF-1α stabilization, and were highly heterogeneous at a cellular level. Frequently, we detected recently reoxygenated cells outside hypoxic areas in the proximity of blood vessels, suggestive of hypoxia-promoted cell migration.

Human elastin-like polypeptides as a versatile platform for exploitation of ultrasensitive bilirubin detection by UnaG.

A new, bifunctional recombinant protein was expressed as the fusion product of human elastin-like polypeptide (HELP) and the bilirubin-binding protein UnaG. The engineered product displays both the HELP-specific property of forming a functional hydrogel matrix and the UnaG-specific capacity of emitting green fluorescence upon ligand binding. The new fusion protein has been proven to be effective at detecting bilirubin in complex environments with high background noise. A cell culture model of the stress response, consisting of bilirubin released in the cell culture medium, was set up to assess the bilirubin-sensing properties of the functional matrix obtained by cross-linking the HELP moiety. Our engineered protein allowed us to monitor cell induction by the release of bilirubin in the culture medium on a nanomolar scale. This study shows that elastin-like protein fusion represents a versatile platform for the development of novel and commercially viable analytical and biosensing devices.

Recovery and Reusability of ApoUnaG Fluorescence Protein from the Unconjugated Bilirubin Complex Structure.

This study is the first report on the separation and reusability of ApoUnaG protein, indicating excellent fluorescence response with high affinity and specificity toward unconjugated bilirubin (UC-BR) molecules, from the UnaG-UC-BR complex structure. The fluorescence properties of the UnaG-UC-BR complex (holo-UnaG) are studied by addition of different metal ions to perform possible interactions with holo-UnaG through absorbance and emission spectra. After addition of metal ions, some changes with respect to the type of metal ions are observed in fluorescence intensity of the holo-UnaG. When compared to metal ions, an excellent quenching response is sighted in the presence of Cu2+ ions by binding with UC-BR in the UnaG-UC-BR complex structure. Obtained non-fluorescence holo-UnaG-Cu2+ complex mixture is passed through Ni-NTA agarose to remove the ingredients such as Cu2+, UC-BR and Cu2+-UC-BR coordination complex from holo-UnaG. From the obtained experiments, it is concluded that Cu2+ ion can be used as an agent for the recovery of ApoUnaG protein via binding with UC-BR molecules. Graphical Abstract Recovery and Reusability of ApoUnaG Fluorescence Protein from the Unconjugated Bilirubin Complex Structure.

An assessment of sensitivity biomarkers for urinary cadmium burden.

BACKGROUND: Excess cadmium (Cd) intake poses a general risk to health and to the kidneys in particular. Among indices of renal dysfunction under Cd burden measures are the urinary N-acetyl-ß-D-glucosidase (UNAG) and urinary ß2-microglobulin (Uß2-MG) enzymes. However, the end-pointed values and the Cd burden threshold remain controversial because the scopes fluctuate widely. METHODS: To ascertain the clinical benchmark dose of urinary Cd (UCd) burden for renal dysfunction, 1595 residents near a Cd site were surveyed. Urine was sampled and assayed. A benchmark dose low (BMDL) was obtained by fitting UCd levels and index levels. RESULTS: We found that over 50% of the subjects were suffering from Cd exposure as their UCd levels far exceeded the national standard threshold of 5.000 µg/g creatinine (cr). Further analysis indicated that Uß2-MG was more sensitive than UNAG for renal dysfunction. The BMDL for UCd was estimated as 3.486 U/g cr (male, where U is unit of enzyme) and 2.998 U/g cr (female) for UNAG. The BMDL for Uß2-MG, which is released into urine from glomerulus after Cd exposure, was found to be 2.506 µg/g cr (male, where µg is the unit of microglobulin) and 2.236 µg/g cr (female). CONCLUSIONS: Uß2-MG is recommended as the sensitivity index for renal dysfunction, with 2.2 µg/g cr as the threshold for clinical diagnosis. Our findings suggest that Uß2-MG is the better biomarker for exposure to Cd.

A dual-ligand-modulable fluorescent protein based on UnaG and calmodulin.

UnaG is a green-emitting fluorescent protein that utilizes unconjugated bilirubin (BR) as its fluorophore. While BR has captured the attention of physiologists as an important antioxidant that scavenges reactive oxygen species in biological membranes, its excessive accumulation causes several clinical symptoms. Although the optimal regulation of BR concentration would result in clinical therapies for aging as well as reduce risks of clinical symptoms, UnaG hardly releases BR owing to its extremely high affinity for BR (Kd = 98 pM). Herein, we engineered the BR binding and fluorescence of UnaG to be Ca2+-sensitive via a genetic insertion of calmodulin (CaM). The resultant UnaG/CaM hybrid protein is a dual-ligand modulable fluorescent protein; binding of the fluorogenic ligand BR is negatively regulated by the other ligand, Ca2+ ion. The affinity for BR differed by three orders of magnitude between the Ca2+-free state (Kd = 9.70 nM) and Ca2+-saturated state (Kd = 9.65 µM). The chimeric protein can release nano- to micro-molar levels of BR with Ca2+ control, and is thus named BReleaCa (BR + releaser + Ca2+). Such a protein hybridization technique will be generally applicable to change the ligand binding properties of a variety of ligand-inducible functional proteins.

Role of UDP-N-acetylmuramic acid in the regulation of MurA activity revealed by molecular dynamics simulations.

The peptidoglycan biosynthesis pathway plays a vital role in bacterial cells, and facilitates peptidoglycan layer formation, a fundamental structural component of the bacterial cell wall. The enzymes in this pathway are candidates for antibiotic development, as most do not have mammalian homologues. The UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase enzyme (MurA) in the peptidoglycan pathway cytoplasmic step is responsible for the phosphoenolpyruvate (PEP)-UNAG catalytic reaction, forming UNAG enolpyruvate and inorganic phosphate. Reportedly, UDP-N-acetylmuramic acid (UNAM) binds tightly to MurA forming a dormant UNAM-PEP-MurA complex and acting as a MurA feedback inhibitor. MurA inhibitors are complex, owing to competitive binding interactions with PEP, UNAM, and UNAG at the MurA active site. We used computational methods to explore UNAM and UNAG binding. UNAM showed stronger hydrogen-bond interactions with the Arg120 and Arg91 residues, which help to stabilize the closed conformation of MurA, than UNAG. Binding free energy calculations using end-point computational methods showed that UNAM has a higher binding affinity than UNAG, when PEP is attached to Cys115. The unbinding process, simulated using τ-random acceleration molecular dynamics, showed that UNAM has a longer relative residence time than UNAG, which is related to several complex dissociation pathways, each with multiple intermediate metastable states. This prevents the loop from opening and exposing the Arg120 residue to accommodate UNAG and potential new ligands. Moreover, we demonstrate the importance of Cys115-linked PEP in closed-state loop stabilization. We provide a basis for evaluating novel UNAM analogues as potential MurA inhibitors. PUBLIC SIGNIFICANCE: MurA is a critical enzyme involved in bacterial cell wall biosynthesis and is involved in antibiotic resistance development. UNAM can remain in the target protein's active site for an extended time compared to its natural substrate, UNAG. The prolonged interaction of this highly stable complex known as the 'dormant complex' comprises UNAM-PEP-MurA and offers insights into antibiotic development, providing potential options against drug-resistant bacteria and advancing our understanding of microbial biology.

Detection of Hypoxia in 2D and 3D Cell Culture Systems Using Genetically Encoded Fluorescent Hypoxia Sensors.

In vivo oxygen availability varies widely between cellular microenvironments, depending on the tissue of origin and its cellular niche. It has long been known that too high or too low oxygen concentrations can act as a biological stressor. Thus, the precise control of oxygen availability should be a consideration for cell culture optimization, especially in the field of three-dimensional (3D) cell culture. In this chapter, we describe a system for visualizing oxygen limitations at a cellular level using human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) that were genetically modified to express a fluorescent hypoxia sensor. This sensor can detect the activation of hypoxia-induced factors (HIF) transcription factors that lead to the expression of the oxygen-independent fluorescent protein, UnaG, at low oxygen concentrations. The response of these hypoxia reporter cells can be evaluated in two-dimensional (2D) and 3D cultivation platforms during exposure to hypoxia (1% O2) and normoxia (21% O2) using fluorescence microscopy and flow cytometry. We show that hypoxia reporter MSCs exhibit a hypoxia-induced fluorescence signal in both 2D and 3D cultivation platforms with fast decay kinetics after reoxygenation, rendering it a valuable tool for studying the cellular microenvironment and regenerative potential of hAD-MSCs in an in vivo-like setting.

Outer Membrane Integrity-Dependent Fluorescence of the Japanese Eel UnaG Protein in Live Escherichia coli Cells.

Reporter genes are important tools in many biological disciplines. The discovery of novel reporter genes is relatively rare. However, known reporter genes are constantly applied to novel applications. This study reports the performance of the bilirubin-dependent fluorescent protein UnaG from the Japanese eel Anguilla japonicas in live Escherichia coli cells in response to the disruption of outer membrane (OM) integrity at low bilirubin (BR) concentrations. Using the E. coli wild-type strain MC4100, its isogenic OM-deficient mutant strain NR698, and different OM-active compounds, we show that BR uptake and UnaG fluorescence depend on a leaky OM at concentrations of 10 µM BR and below, while fluorescence is mostly OM integrity-independent at concentrations above 50 µM BR. We suggest that these properties of the UnaG-BR couple might be applied as a biosensor as an alternative to the OM integrity assays currently in use.

A Fluorescence-Based Quantitative Analysis for Total Bilirubin in Blood and Urine.

BACKGROUND: Bilirubin is a catabolic product of heme metabolism that circulates in the bloodstream in its unconjugated or glucuronide-conjugated form. Because the accumulation of bilirubin in the blood is a common symptom of liver diseases, its measurement in plasma (serum) is important for the diagnosis of these diseases. METHOD: We developed a method to assess total bilirubin levels in serum and urine, using the fluorescent protein UnaG and ß-glucuronidase. RESULTS: Our results indicate good correlation in serum total bilirubin levels between UnaG and the conventional bilirubin oxidase (BOD) methods. We found low levels of conjugated and unconjugated bilirubin in the urine of healthy subject individuals. Urinary bilirubin levels were elevated in patients with liver or bile duct diseases. A simple spot test of bilirubin using serum and urine showed a strong signal in patients with liver diseases. CONCLUSION: The proposed method to assess bilirubin levels in serum and urine will contribute to the accurate diagnosis of health conditions such as jaundice, anemia, and liver disease.

Recent developments on UDP-N-acetylmuramoyl-L-alanine-D-gutamate ligase (Mur D) enzyme for antimicrobial drug development: An emphasis on in-silico approaches.

Introduction: The rapid emergence of antibiotic resistance among various bacterial pathogens has been one of the major concerns of health organizations across the world. In this context, for the development of novel inhibitors against antibiotic-resistant bacterial pathogens, UDP-N-Acetylmuramoyl-L-Alanine-D-Glutamate Ligase (MurD) enzyme represents one of the most apposite targets. Body: The present review focuses on updated advancements on MurD-targeted inhibitors in recent years along with genetic regulation, structural and functional characteristics of the MurD enzyme from various bacterial pathogens. A concise account of various crystal structures of MurD enzyme, submitted into Protein Data Bank is also discussed. Discussion: MurD, an ATP dependent cytoplasmic enzyme is an important target for drug discovery. The genetic organization of MurD enzyme is well elucidated and many crystal structures of MurD enzyme are submitted into Protein Data bank. Various inhibitors against MurD enzyme have been developed so far with an increase in the use of in-silico methods in the recent past. But cell permeability barriers and conformational changes of MurD enzyme during catalytic reaction need to be addressed for effective drug development. So, a combination of in-silico methods along with experimental work is proposed to counter the catalytic machinery of MurD enzyme.

Enhanced UnaG With Minimal Labeling Artifact for Single-Molecule Localization Microscopy.

We introduced enhanced UnaG (eUnaG), a ligand-activatable fluorescent protein, for conventional and super-resolution imaging of subcellular structures in the mammalian cells. eUnaG is a V2L mutant of UnaG with twice brighter bulk fluorescence. We previously discovered the reversible fluorescence switching behavior of UnaG and demonstrated the high photon outputs and high localization numbers in single-molecule localization microscopy (SMLM). In this study, we showed that the fluorescence of eUnaG can be switched off under blue-light illumination, while a high concentration of fluorogenic ligands in the buffer can efficiently restore the fluorescence, as in UnaG. We demonstrated the capacity of eUnaG as an efficient protein label in mammalian cells, as well as for SMLM by utilizing its photoswitchable nature. While cytosolic UnaG proteins showed aggregated patches and fluorescence reduction at high expression levels, eUnaG-labeled protein targets successfully formed their proper structures in mammalian cells without notable distortion from the endogenous structure in the majority of transiently expressing cells. In particular, eUnaG preserved the vimentin filament structures much better than the UnaG. eUnaG provided similarly high single-molecule photon count distribution to UnaG, thus also similarly high resolution in the super-resolution images of various subcellular structures. The sampling coverage analysis of vimentin filaments in SMLM images showed the improvement of labeling efficiency of eUnaG. eUnaG is a high-performance fluorescent protein for fluorescence and single-molecule localization imaging in green emission with minimal labeling artifact.

UnaG as a reporter in adeno-associated virus-mediated gene transfer for biomedical imaging.

Adeno-associated virus (AAV) is one of the most common gene transfer vectors, but it has a limited capacity. A smaller fluorescent protein is urgently needed since it is more suitable to act as a reporter in AAV. In this study, a bilirubin-dependent reporter smaller than EGFP, termed UnaG, was found to have the ability to label the neurons of a mouse brain as clearly as EGFP without the addition of exogenous bilirubin. We also found that UnaG's pH tolerance is better than that of EGFP; however, its fluorescence recovery after protonated quenching is not as good as that of EGFP. In addition, UnaG preserved its fluorescence better than EGFP in SeeDB clearing. Taken together, this study demonstrates that UnaG can act as a small intrinsically fluorescent reporter in the mouse brain without an additional ligand, thus providing an alternative over EGFP for AAV-mediated neuron labeling in mammals.

An Improved HRPE-Based Transcriptional Output Reporter to Detect Hypoxia and Anoxia in Plant Tissue.

Oxygen levels in plant tissues may vary, depending on metabolism, diffusion barriers, and environmental availability. Current techniques to assess the oxic status of plant cells rely primarily on invasive microoptodes or Clark-type electrodes, which are not optimally suited for experiments that require high spatial and temporal resolution. In this case, a genetically encoded oxygen biosensor is required instead. This article reports the design, test, and optimization of a hypoxia-signaling reporter, based on five-time repeated hypoxia-responsive promoter elements (HRPE) driving the expression of different reporter proteins. Specifically, this study aimed to improve its performance as a reporter of hypoxic conditions by testing the effect of different untranslated regions (UTRs) at the 5' end of the reporter coding sequence. Next, we characterized an optimized version of the HRPE promoter (HRPE-Ω) in terms of hypoxia sensitivity and time responsiveness. We also observed that severe oxygen deficiency counteracted the reporter activity due to inhibition of GFP maturation, which requires molecular oxygen. To overcome this limitation, we therefore employed an oxygen-independent UnaG fluorescent protein-coupled to an O2-dependent mCherry fluorophore under the control of the optimized HRPE-Ω promoter. Remarkably, this sensor, provided a different mCherry/UnaG ratiometric output depending on the externally imposed oxygen concentration, providing a solution to distinguish between different degrees of tissue hypoxia. Moreover, a ubiquitously expressed UnaG-mCherry fusion could be used to image oxygen concentrations directly, albeit at a narrow range. The luminescent and fluorescent hypoxia-reporters described here can readily be used to conduct studies that involve anaerobiosis in plants.