T-cell immunity induced and reshaped by an anti-HPV immuno-oncotherapeutic lentiviral vector.

We recently developed an immuno-oncotherapy against human papillomavirus (HPV)-induced tumors based on a lentiviral vector encoding the Early E6 and E7 oncoproteins of HPV16 and HPV18 genotypes, namely "Lenti-HPV-07". The robust and long-lasting anti-tumor efficacy of Lenti-HPV-07 is dependent on CD8+ T-cell induction and remodeling of the tumor microenvironment. Here, we first established that anti-vector immunity induced by Lenti-HPV-07 prime has no impact on the efficacy of a homologous boost to amplify anti-HPV T-cell immunity. To longitudinally monitor the evolution of the T-cell repertoire generated after the prime, homologous or heterologous boost with Lenti-HPV-07, we tracked T-cell clonotypes by deep sequencing of T-Cell Receptor (TCR) variable ß and α chain mRNA, applied to whole peripheral blood cells (PBL) and a T cell population specific of an immunodominant E7HPV16 epitope. We observed a hyper-expansion of clonotypes post prime, accompanied by increased frequencies of HPV-07-specific T cells. Additionally, there was a notable diversification of clonotypes post boost in whole PBL, but not in the E7HPV16-specific T cells. We then demonstrated that the effector functions of such Lenti-HPV-07-induced T cells synergize with anti-checkpoint inhibitory treatments by systemic administration of anti-TIM3 or anti-NKG2A monoclonal antibodies. While Lenti-HPV-07 is about to enter a Phase I/IIa clinical trial, these results will help better elucidate its mode of action in immunotherapy against established HPV-mediated malignancies.

Aging is associated with impaired triggering of TRPV3-mediated cutaneous vasodilation: a crucial process for local heat exposure.

Sensing temperature is vitally important to adapt our body to environmental changes. Local warm detection is required to initiate regulation of cutaneous blood flow, which is part of the peripheral thermoregulatory mechanisms, and thus avoid damage to surrounding tissues. The mechanisms mediating cutaneous vasodilation during local heat stress are impaired with aging. However, the impact of aging on the ability of the skin to detect subtle thermal changes is unknown. Among heat-activated cation channels, transient receptor potential vanilloid 3 (TRPV3) is a thermo-sensor predominantly expressed on keratinocytes and involved in local vascular thermoregulatory mechanisms of the skin in young mice. In the present study, using a murine in vivo model of local heat exposure of the skin, we showed that heat-induced vasodilation was reduced in old mice associated with reduced expression of TRPV3 channels. We also found a decrease in expression and activity of TRPV3 channel, as well as reduced TRPV3-dependent adenosine tri-phosphate release in human primary keratinocytes from old donors. This study shows that aging alters the epidermal TRPV3 channels, which might delay the detection of changes in skin temperature, thereby limiting the mechanisms triggered for local vascular thermoregulation in the old skin.

Full eradication of pre-clinical human papilloma virus-induced tumors by a lentiviral vaccine.

Human papillomavirus (HPV) infections are the cause of all cervical and numerous oropharyngeal and anogenital cancers. The currently available HPV vaccines, which induce neutralizing antibodies, have no therapeutic effect on established tumors. Here, we developed an immuno-oncotherapy against HPV-induced tumors based on a non-integrative lentiviral vector encoding detoxified forms of the Early E6 and E7 oncoproteins of HPV16 and 18 genotypes, namely, "Lenti-HPV-07". A single intramuscular injection of Lenti-HPV-07 into mice bearing established HPV-induced tumors resulted in complete tumor eradication in 100% of the animals and was also effective against lung metastases. This effect correlated with CD8+ T-cell induction and profound remodeling of the tumor microenvironment. In the intra-tumoral infiltrates of vaccinated mice, the presence of large amounts of activated effector, resident memory, and transcription factor T cell factor-1 (TCF-1)+ "stem-like" CD8+ T cells was associated with full tumor eradication. The Lenti-HPV-07-induced immunity was long-lasting and prevented tumor growth after a late re-challenge, mimicking tumor relapse. Lenti-HPV-07 therapy synergizes with an anti-checkpoint inhibitory treatment and therefore shows promise as an immuno-oncotherapy against established HPV-mediated malignancies.

Tailoring confocal microscopy for real-time analysis of photosynthesis at single-cell resolution.

Photoautotrophs' environmental responses have been extensively studied at the organism and ecosystem level. However, less is known about their photosynthesis at the single-cell level. This information is needed to understand photosynthetic acclimation processes, as light changes as it penetrates cells, layers of cells, or organs. Furthermore, cells within the same tissue may behave differently, being at different developmental/physiological stages. Here, we describe an approach for single-cell and subcellular photophysiology based on the customization of confocal microscopy to assess chlorophyll fluorescence quenching by the saturation pulse method. We exploit this setup to (1) reassess the specialization of photosynthetic activities in developing tissues of non-vascular plants; (2) identify a specific subpopulation of phytoplankton cells in marine photosymbiosis, which consolidate energetic connections with their hosts; and (3) examine the link between light penetration and photoprotection responses inside the different tissues that constitute a plant leaf anatomy.

Targeted volume correlative light and electron microscopy of an environmental marine microorganism.

Photosynthetic microalgae are responsible for an important fraction of CO2 fixation and O2 production on Earth. Three-dimensional (3D) ultrastructural characterization of these organisms in their natural environment can contribute to a deeper understanding of their cell biology. However, the low throughput of volume electron microscopy (vEM) methods along with the complexity and heterogeneity of environmental samples pose great technical challenges. In the present study, we used a workflow based on a specific electron microscopy sample preparation method compatible with both light and vEM imaging in order to target one cell among a complex natural community. This method revealed the 3D subcellular landscape of a photosynthetic dinoflagellate, which we identified as Ensiculifera tyrrhenica, with quantitative characterization of multiple organelles. We show that this cell contains a single convoluted chloroplast and show the arrangement of the flagellar apparatus with its associated photosensitive elements. Moreover, we observed partial chromatin unfolding, potentially associated with transcription activity in these organisms, in which chromosomes are permanently condensed. Together with providing insights in dinoflagellate biology, this proof-of-principle study illustrates an efficient tool for the targeted ultrastructural analysis of environmental microorganisms in heterogeneous mixes.

Mice humanized for MHC and hACE2 with high permissiveness to SARS-CoV-2 omicron replication.

Human Angiotensin-Converting Enzyme 2 (hACE2) is the major receptor enabling host cell invasion by SARS-CoV-2 via interaction with Spike. The murine ACE2 does not interact efficiently with SARS-CoV-2 Spike and therefore the laboratory mouse strains are not permissive to SARS-CoV-2 replication. Here, we generated new hACE2 transgenic mice, which harbor the hACE2 gene under the human keratin 18 promoter, in "HHD-DR1" background. HHD-DR1 mice are fully devoid of murine Major Histocompatibility Complex (MHC) molecules of class-I and -II and express only MHC molecules from Human Leukocyte Antigen (HLA) HLA 02.01, DRA01.01, DRB1.01.01 alleles, widely expressed in human populations. We selected three transgenic strains, with various hACE2 mRNA expression levels and distinctive profiles of lung and/or brain permissiveness to SARS-CoV-2 replication. These new hACE2 transgenic strains display high permissiveness to the replication of SARS-CoV-2 Omicron sub-variants, while the previously available B6.K18-ACE22Prlmn/JAX mice have been reported to be poorly susceptible to infection with Omicron. As a first application, one of these MHC- and ACE2-humanized strains was successfully used to show the efficacy of a lentiviral-based COVID-19 vaccine.

Alpha-Lipoic Acid Supplementation Restores Early Age-Related Sensory and Endothelial Dysfunction in the Skin.

Many changes characterize skin aging, and the resulting dysfunctions still constitute a real challenge for our society. The aim of this study was to compare the skin aging of two rat strains, Wistar and Brown Norway (BN), considered as "poorly aging" and "healthy aging" models, respectively, and to assess the effect of alpha-lipoic acid (LPA), especially on skin microcirculation. To this purpose, various skin characteristics were studied at 6, 12, and 24 months and compared to the results of LPA treatment performed at 12 or 24 months. Skin aging occurred in both strains, but we showed an early occurrence of different age-related disorders in the Wistar strain compared to BN strain, especially regarding weight gain, glycemia dysregulation, basal skin perfusion, endothelial function, and skin resistance to low pressure. LPA treatment tended to improve skin resistance to low pressure in BN but not in Wistar despite the improvement of basal skin perfusion, endothelial function, and skin sensory sensitivity. Overall, this study confirmed the healthier aging of BN compared to Wistar strain and the positive effect of LPA on both general state and skin microcirculation.

Intracellular development and impact of a marine eukaryotic parasite on its zombified microalgal host.

Parasites are widespread and diverse in oceanic plankton and many of them infect single-celled algae for survival. How these parasites develop and scavenge energy within the host and how the cellular organization and metabolism of the host is altered remain open questions. Combining quantitative structural and chemical imaging with time-resolved transcriptomics, we unveil dramatic morphological and metabolic changes of the marine parasite Amoebophrya (Syndiniales) during intracellular infection, particularly following engulfment and digestion of nutrient-rich host chromosomes. Changes include a sequential acristate and cristate mitochondrion with a 200-fold increase in volume, a 13-fold increase in nucleus volume, development of Golgi apparatus and a metabolic switch from glycolysis (within the host) to TCA (free-living dinospore). Similar changes are seen in apicomplexan parasites, thus underlining convergent traits driven by metabolic constraints and the infection cycle. In the algal host, energy-producing organelles (plastid, mitochondria) remain relatively intact during most of the infection. We also observed that sugar reserves diminish while lipid droplets increase. Rapid infection of the host nucleus could be a "zombifying" strategy, allowing the parasite to digest nutrient-rich chromosomes and escape cytoplasmic defense, whilst benefiting from maintained carbon-energy production of the host cell.

An intranasal lentiviral booster reinforces the waning mRNA vaccine-induced SARS-CoV-2 immunity that it targets to lung mucosa.

As the coronavirus disease 2019 (COVID-19) pandemic continues and new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern emerge, the adaptive immunity initially induced by the first-generation COVID-19 vaccines starts waning and needs to be strengthened and broadened in specificity. Vaccination by the nasal route induces mucosal, humoral, and cellular immunity at the entry point of SARS-CoV-2 into the host organism and has been shown to be the most effective for reducing viral transmission. The lentiviral vaccination vector (LV) is particularly suitable for this route of immunization owing to its non-cytopathic, non-replicative, and scarcely inflammatory properties. Here, to set up an optimized cross-protective intranasal booster against COVID-19, we generated an LV encoding stabilized spike of SARS-CoV-2 Beta variant (LV::SBeta-2P). mRNA vaccine-primed and -boosted mice, with waning primary humoral immunity at 4 months after vaccination, were boosted intranasally with LV::SBeta-2P. A strong boost effect was detected on cross-sero-neutralizing activity and systemic T cell immunity. In addition, mucosal anti-spike IgG and IgA, lung-resident B cells, and effector memory and resident T cells were efficiently induced, correlating with complete pulmonary protection against the SARS-CoV-2 Delta variant, demonstrating the suitability of the LV::SBeta-2P vaccine candidate as an intranasal booster against COVID-19. LV::SBeta-2P vaccination was also fully protective against Omicron infection of the lungs and central nervous system, in the highly susceptible B6.K18-hACE2IP-THV transgenic mice.

MiR-30a-5p Alters Epidermal Terminal Differentiation during Aging by Regulating BNIP3L/NIX-Dependent Mitophagy.

Chronological aging is characterized by an alteration in the genes' regulatory network. In human skin, epidermal keratinocytes fail to differentiate properly with aging, leading to the weakening of the epidermal function. MiR-30a is particularly overexpressed with epidermal aging, but the downstream molecular mechanisms are still uncovered. The aim of this study was to decipher the effects of miR-30a overexpression in the human epidermis, with a focus on keratinocyte differentiation. We formally identified the mitophagy receptor BNIP3L as a direct target of miR-30a. Using a 3D organotypic model of reconstructed human epidermis overexpressing miR-30a, we observed a strong reduction in BNIP3L expression in the granular layer. In human epidermal sections of skin biopsies from donors of different ages, we observed a similar pattern of BNIP3L decreasing with aging. Moreover, human primary keratinocytes undergoing differentiation in vitro also showed a decreased expression of BNIP3L with age, together with a retention of mitochondria. Moreover, aging is associated with altered mitochondrial metabolism in primary keratinocytes, including decreased ATP-linked respiration. Thus, miR-30a is a negative regulator of programmed mitophagy during keratinocytes terminal differentiation, impairing epidermal homeostasis with aging.

Maintenance of Chronological Aging Features in Culture of Normal Human Dermal Fibroblasts from Old Donors.

Chronological aging is defined as a time-dependent decline of tissue homeostasis which severely impacts skin. Understanding the mechanisms of skin aging is an active research area limited by the lack of relevant in vitro models. Being a component of aging, replicative or stress-induced senescence is repeatedly used to mimic skin aging in vitro, thus presenting only a partial view of the complexity of aging. Herein, we aimed to clarify whether primary normal human dermal fibroblasts retained age-related characteristics when cultured in 2D monolayer, and could be used as a relevant model for aging research. We compared three groups of fibroblasts isolated from different aged donors. We observed strongly decreased population doubling capacities, a reduced clonogenic ability, an impairment in extracellular matrix production together with modifications of respiratory metabolism with an increase in age. These disruptions were particularly marked when comparing fibroblasts isolated from old individuals (over 70 years old) to those isolated from young individuals (18-37 years old), while cells from middle-aged donors exhibited an intermediate profile. These alterations of cell features can be related to the signs of dermis aging, thus showing that cultured primary cells indeed retain some characteristics of the original tissue from which they were extracted.

Full-Lung Prophylaxis against SARS-CoV-2 by One-Shot or Booster Intranasal Lentiviral Vaccination in Syrian Golden Hamsters.

Following the breakthrough of numerous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in recent months and the incomplete efficiency of the currently available vaccines, development of more effective vaccines is desirable. Non-integrative, non-cytopathic and non-inflammatory lentiviral vectors elicit sterilizing prophylaxis against SARS-CoV-2 in preclinical animal models and are particularly suitable for mucosal vaccination, which is acknowledged as the most effective in reducing viral transmission. Here, we demonstrate that a single intranasal administration of a vaccinal lentiviral vector encoding a stabilized form of the original SARS-CoV-2 Spike glycoprotein induces full-lung protection of respiratory tracts and strongly reduces pulmonary inflammation in the susceptible Syrian golden hamster model against the prototype SARS-CoV-2. In addition, we show that a lentiviral vector encoding stabilized Spike of SARS-CoV-2 Beta variant (LV::SBeta-2P) prevents pathology and reduces infectious viral loads in lungs and nasal turbinates following inoculation with the SARS-CoV-2 Omicron variant. Importantly, an intranasal boost with LV::SBeta-2P improves cross-seroneutralization much better in LV::SBeta-2P-primed hamsters than in their counterparts primed with an LV-encoding Spike from the ancestral SARS-CoV-2. These results strongly suggest that an immune imprint with the original Spike sequence has a negative impact on cross-protection against new variants. Our results tackle the issue of vaccine effectiveness in people who have already been vaccinated and have vanished immunity and indicate the efficiency of LV-based intranasal vaccination, either as a single dose or as booster.

Subcellular architecture and metabolic connection in the planktonic photosymbiosis between Collodaria (radiolarians) and their microalgae.

Photosymbiosis is widespread and ecologically important in the oceanic plankton but remains poorly studied. Here, we used multimodal subcellular imaging to investigate the photosymbiosis between colonial Collodaria and their microalga dinoflagellate (Brandtodinium). We showed that this symbiosis is very dynamic whereby symbionts interact with different host cells via extracellular vesicles within the colony. 3D electron microscopy revealed that the photosynthetic apparatus of the microalgae was more voluminous in symbiosis compared to free-living while the mitochondria volume was similar. Stable isotope probing coupled with NanoSIMS showed that carbon and nitrogen were stored in the symbiotic microalga in starch granules and purine crystals respectively. Nitrogen was also allocated to the algal nucleolus. In the host, low 13 C transfer was detected in the Golgi. Metal mapping revealed that intracellular iron concentration was similar in free-living and symbiotic microalgae (c. 40 ppm) and twofold higher in the host, whereas copper concentration increased in symbionts and was detected in the host cell and extracellular vesicles. Sulfur concentration was around two times higher in symbionts (chromatin and pyrenoid) than their host. This study improves our understanding on the functioning of this oceanic photosymbiosis and paves the way for more studies to further assess its biogeochemical significance.

Autophagy and Mitophagy-Related Pathways at the Crossroads of Genetic Pathways Involved in Familial Sarcoidosis and Host-Pathogen Interactions Induced by Coronaviruses.

Sarcoidosis is a multisystem disease characterized by the development and accumulation of granulomas, the hallmark of an inflammatory process induced by environmental and/or infectious and or genetic factors. This auto-inflammatory disease mainly affects the lungs, the gateway to environmental aggressions and viral infections. We have shown previously that genetic predisposition to sarcoidosis occurring in familial cases is related to a large spectrum of pathogenic variants with, however, a clustering around mTOR (mammalian Target Of Rapamycin)-related pathways and autophagy regulation. The context of the COVID-19 pandemic led us to evaluate whether such genetic defects may increase the risk of a severe course of SARS-CoV2 infection in patients with sarcoidosis. We extended a whole exome screening to 13 families predisposed to sarcoidosis and crossed the genes sharing mutations with the list of genes involved in the SARS-CoV2 host-pathogen protein-protein interactome. A similar analysis protocol was applied to a series of 100 healthy individuals. Using ENRICH.R, a comprehensive gene set enrichment web server, we identified the functional pathways represented in the set of genes carrying deleterious mutations and confirmed the overrepresentation of autophagy- and mitophagy-related functions in familial cases of sarcoidosis. The same protocol was applied to the set of genes common to sarcoidosis and the SARS-CoV2-host interactome and found a significant enrichment of genes related to mitochondrial factors involved in autophagy, mitophagy, and RIG-I-like (Retinoic Acid Inducible Gene 1) Receptor antiviral response signaling. From these results, we discuss the hypothesis according to which sarcoidosis is a model for studying genetic abnormalities associated with host response to viral infections as a consequence of defects in autophagy and mitophagy processes.

Cytoklepty in the plankton: A host strategy to optimize the bioenergetic machinery of endosymbiotic algae.

Endosymbioses have shaped the evolutionary trajectory of life and remain ecologically important. Investigating oceanic photosymbioses can illuminate how algal endosymbionts are energetically exploited by their heterotrophic hosts and inform on putative initial steps of plastid acquisition in eukaryotes. By combining three-dimensional subcellular imaging with photophysiology, carbon flux imaging, and transcriptomics, we show that cell division of endosymbionts (Phaeocystis) is blocked within hosts (Acantharia) and that their cellular architecture and bioenergetic machinery are radically altered. Transcriptional evidence indicates that a nutrient-independent mechanism prevents symbiont cell division and decouples nuclear and plastid division. As endosymbiont plastids proliferate, the volume of the photosynthetic machinery volume increases 100-fold in correlation with the expansion of a reticular mitochondrial network in close proximity to plastids. Photosynthetic efficiency tends to increase with cell size, and photon propagation modeling indicates that the networked mitochondrial architecture enhances light capture. This is accompanied by 150-fold higher carbon uptake and up-regulation of genes involved in photosynthesis and carbon fixation, which, in conjunction with a ca.15-fold size increase of pyrenoids demonstrates enhanced primary production in symbiosis. Mass spectrometry imaging revealed major carbon allocation to plastids and transfer to the host cell. As in most photosymbioses, microalgae are contained within a host phagosome (symbiosome), but here, the phagosome invaginates into enlarged microalgal cells, perhaps to optimize metabolic exchange. This observation adds evidence that the algal metamorphosis is irreversible. Hosts, therefore, trigger and benefit from major bioenergetic remodeling of symbiotic microalgae with potential consequences for the oceanic carbon cycle. Unlike other photosymbioses, this interaction represents a so-called cytoklepty, which is a putative initial step toward plastid acquisition.

Morphological bases of phytoplankton energy management and physiological responses unveiled by 3D subcellular imaging.

Eukaryotic phytoplankton have a small global biomass but play major roles in primary production and climate. Despite improved understanding of phytoplankton diversity and evolution, we largely ignore the cellular bases of their environmental plasticity. By comparative 3D morphometric analysis across seven distant phytoplankton taxa, we observe constant volume occupancy by the main organelles and preserved volumetric ratios between plastids and mitochondria. We hypothesise that phytoplankton subcellular topology is modulated by energy-management constraints. Consistent with this, shifting the diatom Phaeodactylum from low to high light enhances photosynthesis and respiration, increases cell-volume occupancy by mitochondria and the plastid CO2-fixing pyrenoid, and boosts plastid-mitochondria contacts. Changes in organelle architectures and interactions also accompany Nannochloropsis acclimation to different trophic lifestyles, along with respiratory and photosynthetic responses. By revealing evolutionarily-conserved topologies of energy-managing organelles, and their role in phytoplankton acclimation, this work deciphers phytoplankton responses at subcellular scales.

[Functional integrity of aging skin, from cutaneous biology to anti-aging strategies]. / Vieillissement et intégrité de la peau - De la biologie cutanée aux stratégies anti-âge.

The skin is a sentinel organ making easily visible the passing of time. Chronological and environmental aging weakens skin structure and functions. The skin barrier, the elastic and mechanical properties of the cutaneous tissue as well as its vascular reactivity are impacted by aging. The barrier dysfunction in aged skin is caused by defects in epidermal keratinocytes renewal and differentiation notably linked to abnormal expression of microRNAs regulating cell death and autophagy. An abnormal balance between synthesis and degradation of matrix proteins modifies the mechanical properties of the dermis in aged skin. Finally, a reduction of the vascular reactivity linked to endothelial dysfunctions is observed in elderly people. These biological processes can be targeted by therapeutic approaches either topical or systemic, especially using anti-oxydants or senolytics. These anti-aging strategies might contribute to restore, at least in part, the functional integrity of aged skin.

TITLE: Vieillissement et intégrité de la peau - De la biologie cutanée aux stratégies anti-âge. ABSTRACT: La peau est un organe sentinelle, soumis au vieillissement chronologique et environnemental qui fragilise sa structure et ses fonctions. La fonction barrière de la peau, ses propriétés élastiques et de résistance, ainsi que sa réactivité vasculaire sont atteintes par le vieillissement dans les compartiments épidermiques, dermiques et vasculaires. Les progrès de la recherche ont permis de révéler des processus biologiques sous-jacents, qui peuvent être ciblés par des approches médicamenteuses topiques ou globales à base notamment d'anti-oxydants ou de sénolytiques. Ces stratégies anti-âge pourront contribuer à restaurer, au moins en partie, l'intégrité fonctionnelle de la peau âgée.

Nucleo-plastidic PAP8/pTAC6 couples chloroplast formation with photomorphogenesis.

The initial greening of angiosperms involves light activation of photoreceptors that trigger photomorphogenesis, followed by the development of chloroplasts. In these semi-autonomous organelles, construction of the photosynthetic apparatus depends on the coordination of nuclear and plastid gene expression. Here, we show that the expression of PAP8, an essential subunit of the plastid-encoded RNA polymerase (PEP) in Arabidopsis thaliana, is under the control of a regulatory element recognized by the photomorphogenic factor HY5. PAP8 protein is localized and active in both plastids and the nucleus, and particularly required for the formation of late photobodies. In the pap8 albino mutant, phytochrome-mediated signalling is altered, degradation of the chloroplast development repressors PIF1/PIF3 is disrupted, HY5 is not stabilized, and the expression of the photomorphogenesis regulator GLK1 is impaired. PAP8 translocates into plastids via its targeting pre-sequence, interacts with the PEP and eventually reaches the nucleus, where it can interact with another PEP subunit pTAC12/HMR/PAP5. Since PAP8 is required for the phytochrome B-mediated signalling cascade and the reshaping of the PEP activity, it may coordinate nuclear gene expression with PEP-driven chloroplastic gene expression during chloroplast biogenesis.

In vivo characterization of the activities of novel cyclodipeptide oxidases: new tools for increasing chemical diversity of bioproduced 2,5-diketopiperazines in Escherichia coli.

BACKGROUND: Cyclodipeptide oxidases (CDOs) are enzymes involved in the biosynthesis of 2,5-diketopiperazines, a class of naturally occurring compounds with a large range of pharmaceutical activities. CDOs belong to cyclodipeptide synthase (CDPS)-dependent pathways, in which they play an early role in the chemical diversification of cyclodipeptides by introducing Cα-Cß dehydrogenations. Although the activities of more than 100 CDPSs have been determined, the activities of only a few CDOs have been characterized. Furthermore, the assessment of the CDO activities on chemically-synthesized cyclodipeptides has shown these enzymes to be relatively promiscuous, making them interesting tools for cyclodipeptide chemical diversification. The purpose of this study is to provide the first completely microbial toolkit for the efficient bioproduction of a variety of dehydrogenated 2,5-diketopiperazines. RESULTS: We mined genomes for CDOs encoded in biosynthetic gene clusters of CDPS-dependent pathways and selected several for characterization. We co-expressed each with their associated CDPS in the pathway using Escherichia coli as a chassis and showed that the cyclodipeptides and the dehydrogenated derivatives were produced in the culture supernatants. We determined the biological activities of the six novel CDOs by solving the chemical structures of the biologically produced dehydrogenated cyclodipeptides. Then, we assessed the six novel CDOs plus two previously characterized CDOs in combinatorial engineering experiments in E. coli. We co-expressed each of the eight CDOs with each of 18 CDPSs selected for the diversity of cyclodipeptides they synthesize. We detected more than 50 dehydrogenated cyclodipeptides and determined the best CDPS/CDO combinations to optimize the production of 23. CONCLUSIONS: Our study establishes the usefulness of CDPS and CDO for the bioproduction of dehydrogenated cyclodipeptides. It constitutes the first step toward the bioproduction of more complex and diverse 2,5-diketopiperazines.

Retrograde signals from mitochondria reprogramme skoto-morphogenesis in Arabidopsis thaliana via alternative oxidase 1a.

The early steps in germination and development of angiosperm seedlings often occur in the dark, inducing a special developmental programme called skoto-morphogenesis. Under these conditions photosynthesis cannot work and all energetic requirements must be fulfilled by mitochondrial metabolization of storage energies. Here, we report the physiological impact of mitochondrial dysfunctions on the skoto-morphogenic programme by using the Arabidopsis rpoTmp mutant. This mutant is defective in the T7-phage-type organellar RNA polymerase shared by plastids and mitochondria. Lack of this enzyme causes a mitochondrial dysfunction resulting in a strongly reduced mitochondrial respiratory chain and a compensatory upregulation of the alternative-oxidase (AOX)-dependent respiration. Surprisingly, the mutant exhibits a triple-response-like phenotype with a twisted apical hook and a shortened hypocotyl. Highly similar phenotypes were detected in other respiration mutants (rug3 and atphb3) and in WT seedlings treated with the respiration inhibitor KCN. Further genetic and molecular data suggest that the observed skoto-morphogenic alterations are specifically dependent on the activity of the AOX1a enzyme. Microarray analyses indicated that a retrograde signal from mitochondria activates the ANAC017-dependent pathway which controls the activation of AOX1A transcription. In sum, our analysis identifies AOX as a functional link that couples the formation of a triple-response-like phenotype to mitochondrial dysfunction. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.