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Oxygen deprivation induces multiple changes at the cellular and organismal levels, and its re-supply also brings another special physiological status. We have investigated the effects of hypoxia/re-oxygenation on embryonic growth using the zebrafish model: hypoxia slows embryonic growth, but re-oxygenation induces growth spurt or  catch-up   growth . The mitogen-activated kinase (MAPK)-pathway downstream insulin-like growth factor (IGF/Igf) has been revealed to positively regulate the re-oxygenation-induced catch-up growth, and the role of reactive oxygen species generated by environmental oxygen fluctuation is potentially involved in the phenomenon. Here, we report the role of NADPH-oxidase (Nox)-dependent hydrogen peroxide (H 2 O 2 ) production in the MAPK-activation and catch-up growth. The inhibition of Nox significantly blunted catch-up growth and MAPK-activity. Amongst two zebrafish insulin receptor substrate 2 genes ( irs2a and irs2b ), the loss of  irs2b , but not its paralog  irs2a , resulted in blunted MAPK-activation and catch-up growth. Furthermore,  irs2b  forcedly expressed in mammalian cells allowed IGF-MAPK augmentation in the presence of H 2 O 2 , and the irs2b  deficiency completely abolished the somatotropic action of Nox in re-oxygenation condition. These results indicate that redox signaling alters IGF/Igf signaling to facilitate hypoxia/re-oxygenation-induced embryonic growth compensation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zasu, Hishima, Thauvin, Yoneyama, Kitani, Hakuno, Volovitch, Takahashi, Vriz, Rampon and Kamei.)

Among molecules that bridge environment, cell metabolism, and cell signaling, hydrogen peroxide (H 2 O 2 ) recently appeared as an emerging but central player. Its level depends on cell metabolism and environment and was recently shown to play key roles during embryogenesis, contrasting with its long-established role in disease progression. We decided to explore whether the secreted morphogen Sonic hedgehog (Shh), known to be essential in a variety of biological processes ranging from embryonic development to adult tissue homeostasis and cancers, was part of these interactions. Here, we report that H 2 O 2 levels control key steps of Shh delivery in cell culture: increased levels reduce primary secretion, stimulate endocytosis and accelerate delivery to recipient cells; in addition, physiological in vivo modulation of H 2 O 2 levels changes Shh distribution and tissue patterning. Moreover, a feedback loop exists in which Shh trafficking controls H 2 O 2 synthesis via a non-canonical BOC-Rac1 pathway, leading to cytoneme growth. Our findings reveal that Shh directly impacts its own distribution, thus providing a molecular explanation for the robustness of morphogenesis to both environmental insults and individual variability.

Reactive oxygen species (ROS), originally classified as toxic molecules, have attracted increasing interest given their actions in cell signaling. Hydrogen peroxide (H2O2), the major ROS produced by cells, acts as a second messenger to modify redox-sensitive proteins or lipids. After caudal fin amputation, tight spatiotemporal regulation of ROS is required first for wound healing and later to initiate the regenerative program. However, the mechanisms carrying out this sustained ROS production and their integration with signaling pathways remain poorly understood. We focused on the early dialog between H2O2 and Sonic hedgehog (Shh) during zebrafish fin regeneration. We demonstrate that H2O2 controls Shh expression and that Shh in turn regulates the H2O2 level via a canonical pathway. Moreover, the means of this tight reciprocal control change during the successive phases of the regenerative program. Dysregulation of the Hedgehog pathway has been implicated in several developmental syndromes, diabetes and cancer. These data support the existence of an early positive crosstalk between Shh and H2O2 that might be more generally involved in various processes paving the way to improve regenerative processes, particularly in vertebrates., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The lack of tools to monitor the dynamics of (pseudo)hypohalous acids in live cells and tissues hinders a better understanding of inflammatory processes. Here we present a fluorescent genetically encoded biosensor, Hypocrates, for the visualization of (pseudo)hypohalous acids and their derivatives. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from Escherichia coli. We show that Hypocrates is ratiometric, reversible, and responds to its analytes in the 10 6  M -1 s -1 range. Solving the Hypocrates X-ray structure provided insights into its sensing mechanism, allowing determination of the spatial organization in this circularly permuted fluorescent protein-based redox probe. We exemplify its applicability by imaging hypohalous stress in bacteria phagocytosed by primary neutrophils. Finally, we demonstrate that Hypocrates can be utilized in combination with HyPerRed for the simultaneous visualization of (pseudo)hypohalous acids and hydrogen peroxide dynamics in a zebrafish tail fin injury model., (© 2022. The Author(s).)

Catalases (CAT) are antioxidant metalloenzymes necessary for life in oxygen-metabolizing cells to regulate H 2 O 2 concentration by accelerating its dismutation. Many physiopathological situations are associated with oxidative stress resulting from H 2 O 2 overproduction, during which antioxidant defenses are overwhelmed. We have used a combinatorial approach associated with an activity-based screening to discover a first peptidyl di-copper complex mimicking CAT. The complex was studied in detail and characterized for its CAT activity both in solutions and in cells using different analytical methods. The complex exhibited CAT activity in solutions and, more interestingly, on HyPer HeLa cells that possess a genetically encoded ratiometric fluorescent sensors of H 2 O 2 . These results highlight the efficiency of a combinatorial approach for the discovery of peptidyl complexes that exhibit catalytic activity.

Oxidative stress that results from an imbalance between the concentrations of reactive species (RS) and antioxidant defenses is associated with many pathologies. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase are among the key enzymes that maintain the low nanomolar physiological concentrations of superoxide and hydrogen peroxide. The increase in the levels of these species and their progeny could have deleterious effects. In this context, chemists have developed SOD and CAT mimics to supplement them when cells are overwhelmed with oxidative stress. However, the beneficial activity of such molecules in cells depends not only on their intrinsic catalytic activities but also on their stability in biological context, their cell penetration and their cellular localization. We have employed cellular assays to characterize several compounds that possess SOD and CAT activities and have been frequently used in cellular and animal models. We used cellular assays that address SOD and CAT activities of the compounds. Finally, we determined the effect of compounds on the suppression of the inflammation in HT29-MD2 cells challenged by lipopolysaccharide. When the assay requires penetration inside cells, the SOD mimics Mn(III) meso-tetrakis(N-(2'-n-butoxyethyl)pyridinium-2-yl)porphyrin (MnTnBuOE-2-PyP 5+ ) and Mn(II) dichloro[(4aR,13aR,17aR,21aR)-1,2,3,4,4a,5,6,12,13,13a,14,15,16,17,17a,18,19,20,21,21a-eicosahydro-11,7-nitrilo-7Hdibenzo[b,h] [1,4, 7,10] tetraazacycloheptadecine-κN5,κN13,κN18,κN21,κN22] (Imisopasem manganese, M40403, CG4419) were found efficacious at 10 μM, while Mn(II) chloro N-(phenolato)-N,N'-bis[2-(N-methyl-imidazolyl)methyl]-ethane-1,2-diamine (Mn1) requires an incubation at 100 μM. This study thus demonstrates that MnTnBuOE-2-PyP 5+ , M40403 and Mn1 were efficacious in suppressing inflammatory response in HT29-MD2 cells and such action appears to be related to their ability to enter the cells and modulate reactive oxygen species (ROS) levels., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Spectrally separated fluorophores allow the observation of multiple targets simultaneously inside living cells, leading to a deeper understanding of the molecular interplay that regulates cell function and fate. Chemogenetic systems combining a tag and a synthetic fluorophore provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. Here, we present the engineering of a set of spectrally orthogonal fluorogen-activating tags based on the fluorescence-activating and absorption shifting tag (FAST) that are compatible with two-color, live-cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. This pair of orthogonal tags allowed the creation of a two-color cell cycle sensor capable of detecting very short, early cell cycles in zebrafish development and the development of split complementation systems capable of detecting multiple protein-protein interactions by live-cell fluorescence microscopy.

Observing the localization, the concentration, and the distribution of proteins in cells or organisms is essential to understand theirs functions. General and versatile methods allowing multiplexed imaging of proteins under a large variety of experimental conditions are thus essential for deciphering the inner workings of cells and organisms. Here, we present a general method based on the non-covalent labeling of a small protein tag, named FAST (fluorescence-activating and absorption-shifting tag), with various fluorogenic ligands that light up upon labeling, which makes the simple, robust, and versatile on-demand labeling of fusion proteins in a wide range of experimental systems possible., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Far-red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we describe the generation of frFAST (far-red Fluorescence Activating and absorption Shifting Tag), a 14-kDa monomeric protein that forms a bright far-red fluorescent assembly with (4-hydroxy-3-methoxy-phenyl)allylidene rhodanine (HPAR-3OM). As HPAR-3OM is essentially non-fluorescent in solution and in cells, frFAST can be imaged with high contrast in presence of free HPAR-3OM, which allowed the rapid and efficient imaging of frFAST fusions in live cells, zebrafish embryo/larvae, and chicken embryos. Beyond enabling the genetic encoding of far-red fluorescence, frFAST allowed the design of a far-red chemogenetic reporter of protein-protein interactions, demonstrating its great potential for the design of innovative far-red emitting biosensors., (© 2020 Wiley-VCH GmbH.)

Although a physiological role for redox signaling is now clearly established, the processes sensitive to redox signaling remains to be identified. Ratiometric probes selective for H 2 O 2 have revealed its complex spatiotemporal dynamics during neural development and adult regeneration and perturbations of H 2 O 2 levels disturb cell plasticity and morphogenesis. Here we ask whether endogenous H 2 O 2 could participate in the patterning of the embryo. We find that perturbations of endogenous H 2 O 2 levels impact on the distribution of the Engrailed homeoprotein, a strong determinant of midbrain patterning. Engrailed 2 is secreted from cells with high H 2 O 2 levels and taken up by cells with low H 2 O 2 levels where it leads to increased H 2 O 2 production, steering the directional spread of the Engrailed gradient. These results illustrate the interplay between protein signaling pathways and metabolic processes during morphogenetic events.

Hydrogen peroxide (H 2 O 2 ) is a key redox intermediate generated within cells. Existing probes for H 2 O 2 have not solved the problem of detection of the ultra-low concentrations of the oxidant: these reporters are not sensitive enough, or pH-dependent, or insufficiently bright, or not functional in mammalian cells, or have poor dynamic range. Here we present HyPer7, the first bright, pH-stable, ultrafast, and ultrasensitive ratiometric H 2 O 2 probe. HyPer7 is fully functional in mammalian cells and in other higher eukaryotes. The probe consists of a circularly permuted GFP integrated into the ultrasensitive OxyR domain from Neisseria meningitidis. Using HyPer7, we were able to uncover the details of H 2 O 2 diffusion from the mitochondrial matrix, to find a functional output of H 2 O 2 gradients in polarized cells, and to prove the existence of H 2 O 2 gradients in wounded tissue in vivo. Overall, HyPer7 is a probe of choice for real-time H 2 O 2 imaging in various biological contexts., Competing Interests: Declaration of Interests The authors declare no competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Reactive oxygen species (ROS) and downstream products of lipid oxidation are emerging as important secondary messengers in tissue homeostasis. However, their regulation and mechanism of action remain poorly studied in vivo during normal development. Here, we reveal that the fine regulation of hydrogen peroxide (H 2 O 2 ) levels by its scavenger Catalase to mediate the switch from proliferation to differentiation in retinal progenitor cells (RPCs) is crucial. We identify 9-hydroxystearic acid (9-HSA), an endogenous downstream lipid peroxidation product, as a mediator of this effect in the zebrafish retina. We show that the 9-HSA proliferative effect is due to the activation of Notch and Wnt pathways through the inhibition of the histone deacetylase 1. We show that the local and temporal manipulation of H 2 O 2 levels in RPCs is sufficient to trigger their premature differentiation. We finally propose a mechanism that links H 2 O 2 homeostasis and neuronal differentiation via the modulation of lipid peroxidation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The tight control of reactive oxygen species (ROS) levels is required during regeneration. H 2 O 2 in particular assumes clear signalling functions at different steps in this process. Injured nerves induce high levels of H 2 O 2 through the activation of the Hedgehog (Shh) pathway, providing an environment that promotes cell plasticity, progenitor recruitment and blastema formation. In turn, high H 2 O 2 levels contribute to growing axon attraction. Once re-innervation is completed, nerves subsequently downregulate H 2 O 2 levels to their original state. A similar regulatory loop between H 2 O 2 levels and nerves also exists during development. This suggests that redox signalling is a major actor in cell plasticity., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A novel selective androgen receptor modulator scaffold has been discovered through structural modifications of hydantoin antiandrogens. Several 4-(4-hydroxyphenyl)-N-arylhydantoins displayed partial agonism with nanomolar in vitro potency in transactivation experiments using androgen receptor (AR) transfected cells. In a standard castrated male rat model, several compounds showed good anabolic activity on levator ani muscle, dissociated from the androgenic activity on ventral prostate, after oral dosing at 30 mg/kg. (+)-4-[3,4-Dimethyl-2,5-dioxo-4-(4-hydroxyphenyl)imidazolidin-1-yl]-2-(trifluoromethyl)benzonitrile ((+)-11b) displayed anabolic potency with a strong dissociation between levator ani muscle and ventral prostate (A(50) = 0.5 mg/kg vs 70 mg/kg). The binding modes of two compounds, including (+)-11b, within the AR ligand-binding domain have been studied by cocrystallization experiments using a coactivator-like peptide. Both compounds bound to the same site, and the overall structures of the AR were very similar.

Pharmaceutical products, including capsules, oral suspensions, and solutions, are prepared by hospital pharmacists if no commercial product is available. Identification of the raw materials is a regulatory requirement before manufacturing (compounding). Because of the standard methods used, however, this is often time-consuming and laborious in a hospital setting. This article describes the use of near infrared spectroscopy in combination with chemometric methods for discrimination of raw materials. Sixty-three pulverized powder samples were discriminated by using reference samples (identity guaranteed by supplier and confirmed by mid infrared analyses) and NIRCal cluster analyses. A routine expert application involving optimized calibrations (n=6) was developed, which allowed a rapid and nondestructive release procedure for every powder-based raw material received. This technique is superior to established identification analyses because of reduced quarantine times and cost savings.

The ligand-binding domain of the human androgen receptor has been cloned, overproduced and crystallized in the presence of a coactivator-like 11-mer peptide and two different nonsteroidal ligands. The crystals of the two ternary complexes were isomorphous and belonged to space group P2(1)2(1)2(1), with one molecule in the asymmetric unit. They diffracted to 1.7 and 1.95 A resolution, respectively. Structure determination of these two complexes will help in understanding the mode of binding of selective nonsteroidal androgens versus endogenous steroidal ligands and possibly the origin of their tissue selectivity.

Little is known about the natural history and the pathogenicity of the TT virus (TTV). We present our findings of a cross-sectional study based on the TTV DNA screening of 173 multiple-transfused patients and a longitudinal study based on the follow-up of TTV DNA-positive patients. Overall, 48 patients (27.7%) tested positive for TTV DNA. The influence of the number of blood donor exposures on the prevalence of blood-borne viral infection indicates that TTV, hepatitis C virus (HCV), and an RNA virus known as GB virus C/hepatitis G virus (GBV-C/HGV) share a parenteral transmission, but that TTV, in contrast to the 2 other viruses, is also transmitted by at least another efficient means. The patients having a well-defined date of TTV infection were positive for TTV DNA during a mean period of 3.1 years. A chronic infection was observed in 31 cases (86%). TTV carriage appeared clinically benign in all patients. No clinical evidence of a disease potentially linked to the TTV infection was observed in patients with TTV DNA carriage over several years. The majority of TTV carriers had no biochemical evidence of liver disease. The prevalence of elevated serum alanine aminotransferase (ALT) level was higher in the TTV DNA-positive group, even in the absence of HCV infection, but the observed peaks of ALT level were most often transient and very mild. The prevalence of TTV DNA observed in blood recipients is consistent with that of TTV infection observed in blood donors. TTV infection frequently tends to persist. (Blood. 2000;95:347-351)

To clarify the frequency and prognostic significance of a plasma human immunodeficiency virus (HIV) RNA load below the detection threshold during the natural history of infection, an ultrasensitive assay was used to identify persons with low virus loads in a cohort of 111 untreated subjects with a known date of seroconversion. Six persons had HIV RNA loads <40 viral copies (VC)/mL during the first years of HIV infection. The probability of meeting the criteria for long-term nonprogression was higher in these subjects (P=.043). However, a virus load <40 VC/mL was rare during the natural history of infection, even during the first years of symptomless HIV carriage. Such data confirm the general trend of disease progression in the entire population of HIV carriers.

The prevalence of GB virus C (GBV-C) infection is high in human immunodeficiency virus (HIV)-infected persons. However, the long-term consequences of coinfection are unknown. HIV-positive persons with a well-defined duration of infection were screened on the basis of their GBV-C/hepatitis G virus (HGV) RNA status and studied. GBV-C/HGV viremia was observed in 23, who carried the virus over a mean of 7.7 years. All parameters (survival, CDC stage B/C, HIV RNA load, CD4 T cell count) showed significant differences in terms of the cumulative progression rate between persons positive and negative for GBV-C/HGV RNA. When GBV-C/HGV RNA-positive and -unexposed subjects were matched by age, sex, baseline HIV RNA load, and baseline CD4 T cell count, HIV disease progression appeared worse in GBV-C/HGV RNA-negative subjects. The carriage of GBV-C/HGV RNA is associated with a slower progression of HIV disease in coinfected persons.

A cohort of 103 human immunodeficiency virus type 1 (HIV-1)-infected persons with well-defined dates of seroconversion were studied to determine whether baseline plasma HIV RNA loads 6-12 months after seroconversion have prognostic value. Baseline plasma virus loads had predictive value for the disease-free survival rate and for the survival rate. The level of baseline HIV RNA also had a strong negative predictive value for the CD4+ T cell count during the fifth year of infection: A baseline load >5 log was predictive of a CD4+ T cell count <500/mm3 5 years after infection. Baseline HIV RNA load was a CD4+ T cell-independent predictor of progression to death. The major finding was that the disease outcome for HIV-1-infected persons is already determined during the first year of infection.