A safe and potentiated multi-type HPV L2-E7 nanoparticle vaccine with combined prophylactic and therapeutic activity.

Persistent infection with high-risk human papillomavirus (HPV) is widely recognized as the primary cause of cervical and other malignant cancers. There are six licensed prophylactic vaccines available against HPV, but none of them shows any significant therapeutic effect on pre-existing infections or lesions. Thus, a prophylactic vaccine also endowed with therapeutic activity would afford protection regardless of the vaccine recipients HPV-infection status. Here, we describe the refinement and further potentiation of a dual-purpose HPV nanoparticle vaccine (hereafter referred to as cPANHPVAX) relying on eight different HPV L2 peptide epitopes and on the E7 oncoantigens from HPV16 and 18. cPANHPVAX not only induces anti-HPV16 E7 cytotoxic T-cell responses in C57BL/6 mice, but also anti-HPV18 E7 T-cell responses in transgenic mice with the A2.DR1 haplotype. These cytotoxic responses add up to a potent, broad-coverage humoral (HPV-neutralizing) response. cPANHPVAX safety was further improved by deletion of the pRb-binding domains of E7. Our dual-purpose vaccine holds great potential for clinical translation as an immune-treatment capable of targeting active infections as well as established HPV-related malignancies, thus benefiting both uninfected and infected individuals.

Inter-epitope spacer variation within polytopic L2-based human papillomavirus antigens affects immunogenicity.

The human papillomavirus minor capsid protein L2 is being extensively explored in pre-clinical studies as an attractive vaccine antigen capable of inducing broad-spectrum prophylactic antibody responses. Recently, we have developed two HPV vaccine antigens - PANHPVAX and CUT-PANHPVAX- both based on heptameric nanoparticle antigens displaying polytopes of the L2 major cross-neutralizing epitopes of eight mucosal and twelve cutaneous HPV types, respectively. Prompted by the variable neutralizing antibody responses against some of the HPV types targeted by the antigens observed in previous studies, here we investigated the influence on immunogenicity of six distinct glycine-proline spacers inserted upstream to a specific L2 epitope. We show that spacer variants differentially influence antigen immunogenicity in a mouse model, with the antigen constructs M8merV6 and C12merV6 displaying a superior ability in the induction of neutralizing antibodies as determined by pseudovirus-based neutralization assays (PBNAs). L2-peptide enzyme-linked immunosorbent assay (ELISA) assessments determined the total anti-L2 antibody level for each antigen variant, showing for the majority of sera a correlation with their repective neutralizing antibody level. Surface Plasmon Resonance revealed that L2 epitope-specific, neutralizing monoclonal antibodies (mAbs) display distinct avidities to different antigen spacer variants. Furthermore, mAb affinity toward individual spacer variants was well correlated with their neutralizing antibody induction capacity, indicating that the mAb affinity assay predicts L2-based antigen immunogenicity. These observations provide insights on the development and optimization of L2-based HPV vaccines.

A dry powder formulation for peripheral lung delivery and absorption of an anti-SARS-CoV-2 ACE2 decoy polypeptide.

One of the strategies proposed for the neutralization of SARS-CoV-2 has been to synthetize small proteins able to act as a decoy towards the virus spike protein, preventing it from entering the host cells. In this work, the incorporation of one of these proteins, LCB1, within a spray-dried formulation for inhalation was investigated. A design of experiments approach was applied to investigate the optimal condition for the manufacturing of an inhalable powder. The lead formulation, containing 6% w/w of LCB1 as well as trehalose and L-leucine as excipients, preserved the physical stability of the protein and its ability to neutralize the virus. In addition, the powder had a fine particle fraction of 58.6% and a very high extra-fine particle fraction (31.3%) which could allow a peripheral deposition in the lung. The in vivo administration of the LCB1 inhalation powder showed no significant difference in the pharmacokinetic from the liquid formulation, indicating the rapid dissolution of the microparticles and the protein capability to translocate into the plasma. Moreover, LCB1 in plasma samples still maintained the ability to neutralize the virus. In conclusion, the optimized spray drying conditions allowed to obtain an inhalation powder able to preserve the protein biological activity, rendering it suitable for a systemic prevention of the viral infection via pulmonary administration.

Time-course transcriptome analysis of a double challenge bleomycin-induced lung fibrosis rat model uncovers ECM homoeostasis-related translationally relevant genes.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is an irreversible disorder with a poor prognosis. The incomplete understanding of IPF pathogenesis and the lack of accurate animal models is limiting the development of effective treatments. Thus, the selection of clinically relevant animal models endowed with similarities with the human disease in terms of lung anatomy, cell biology, pathways involved and genetics is essential. The bleomycin (BLM) intratracheal murine model is the most commonly used preclinical assay to evaluate new potential therapies for IPF. Here, we present the findings derived from an integrated histomorphometric and transcriptomic analysis to investigate the development of lung fibrosis in a time-course study in a BLM rat model and to evaluate its translational value in relation to IPF. METHODS: Rats were intratracheally injected with a double dose of BLM (days 0-4) and sacrificed at days 7, 14, 21, 28 and 56. Histomorphometric analysis of lung fibrosis was performed on left lung sections. Transcriptome profiling by RNAseq was performed on the right lung lobes and results were compared with nine independent human gene-expression IPF studies. RESULTS: The histomorphometric and transcriptomic analyses provided a detailed overview in terms of temporal gene-expression regulation during the establishment and repair of the fibrotic lesions. Moreover, the transcriptomic analysis identified three clusters of differentially coregulated genes whose expression was modulated in a time-dependent manner in response to BLM. One of these clusters, centred on extracellular matrix (ECM)-related process, was significantly correlated with histological parameters and gene sets derived from human IPF studies. CONCLUSIONS: The model of lung fibrosis presented in this study lends itself as a valuable tool for preclinical efficacy evaluation of new potential drug candidates. The main finding was the identification of a group of persistently dysregulated genes, mostly related to ECM homoeostasis, which are shared with human IPF.

Natural heteroclitic-like peptides are generated by SARS-CoV-2 mutations.

Humoral immunity is sensitive to evasion by SARS-CoV-2 mutants, but CD8 T cells seem to be more resistant to mutational inactivation. By a systematic analysis of 30 spike variant peptides containing the most relevant VOC and VOI mutations that have accumulated overtime, we show that in vaccinated and convalescent subjects, mutated epitopes can have not only a neutral or inhibitory effect on CD8 T cell recognition but can also enhance or generate de novo CD8 T cell responses. The emergence of these mutated T cell function enhancing epitopes likely reflects an epiphenomenon of SARS-CoV-2 evolution driven by antibody evasion and increased virus transmissibility. In a subset of individuals with weak and narrowly focused CD8 T cell responses selection of these heteroclitic-like epitopes may bear clinical relevance by improving antiviral protection. The functional enhancing effect of these peptides is also worth of consideration for the future development of new generation, more potent COVID-19 vaccines.

Time-resolved transcriptomic profiling of the developing rabbit's lungs: impact of premature birth and implications for modelling bronchopulmonary dysplasia.

BACKGROUND: Premature birth, perinatal inflammation, and life-saving therapies such as postnatal oxygen and mechanical ventilation are strongly associated with the development of bronchopulmonary dysplasia (BPD); these risk factors, alone or combined, cause lung inflammation and alter programmed molecular patterns of normal lung development. The current knowledge on the molecular regulation of lung development mainly derives from mechanistic studies conducted in newborn rodents exposed to postnatal hyperoxia, which have been proven useful but have some limitations. METHODS: Here, we used the rabbit model of BPD as a cost-effective alternative model that mirrors human lung development and, in addition, enables investigating the impact of premature birth per se on the pathophysiology of BPD without further perinatal insults (e.g., hyperoxia, LPS-induced inflammation). First, we characterized the rabbit's normal lung development along the distinct stages (i.e., pseudoglandular, canalicular, saccular, and alveolar phases) using histological, transcriptomic and proteomic analyses. Then, the impact of premature birth was investigated, comparing the sequential transcriptomic profiles of preterm rabbits obtained at different time intervals during their first week of postnatal life with those from age-matched term pups. RESULTS: Histological findings showed stage-specific morphological features of the developing rabbit's lung and validated the selected time intervals for the transcriptomic profiling. Cell cycle and embryo development, oxidative phosphorylation, and WNT signaling, among others, showed high gene expression in the pseudoglandular phase. Autophagy, epithelial morphogenesis, response to transforming growth factor ß, angiogenesis, epithelium/endothelial cells development, and epithelium/endothelial cells migration pathways appeared upregulated from the 28th day of gestation (early saccular phase), which represents the starting point of the premature rabbit model. Premature birth caused a significant dysregulation of the inflammatory response. TNF-responsive, NF-κB regulated genes were significantly upregulated at premature delivery and triggered downstream inflammatory pathways such as leukocyte activation and cytokine signaling, which persisted upregulated during the first week of life. Preterm birth also dysregulated relevant pathways for normal lung development, such as blood vessel morphogenesis and epithelial-mesenchymal transition. CONCLUSION: These findings establish the 28-day gestation premature rabbit as a suitable model for mechanistic and pharmacological studies in the context of BPD.

An Optimized Workflow for the Discovery of New Antimicrobial Compounds Targeting Bacterial RNA Polymerase Complex Formation.

Bacterial resistance represents a major health problem worldwide and there is an urgent need to develop first-in-class compounds directed against new therapeutic targets. We previously developed a drug-discovery platform to identify new antimicrobials able to disrupt the protein-protein interaction between the ß' subunit and the σ70 initiation factor of bacterial RNA polymerase, which is essential for transcription. As a follow-up to such work, we have improved the discovery strategy to make it less time-consuming and more cost-effective. This involves three sequential assays, easily scalable to a high-throughput format, and a subsequent in-depth characterization only limited to hits that passed the three tests. This optimized workflow, applied to the screening of 5360 small molecules from three synthetic and natural compound libraries, led to the identification of six compounds interfering with the ß'-σ70 interaction, and thus was capable of inhibiting promoter-specific RNA transcription and bacterial growth. Upon supplementation with a permeability adjuvant, the two most potent transcription-inhibiting compounds displayed a strong antibacterial activity against Escherichia coli with minimum inhibitory concentration (MIC) values among the lowest (0.87-1.56 µM) thus far reported for ß'-σ PPI inhibitors. The newly identified hit compounds share structural feature similarities with those of a pharmacophore model previously developed from known inhibitors.

A broadly protective vaccine against cutaneous human papillomaviruses.

Skin colonization by human papillomavirus (HPV) is typically related to inconspicuous cutaneous infections without major disease or complications in immunocompetent individuals. However, in immunosuppressed patients, especially organ transplanted recipients, cutaneous HPV infections may cause massive, highly spreading and recurrent skin lesions upon synergism with UV-exposure. Current HPV prophylactic vaccines are not effective against cutaneous HPV types (cHPV). By applying a modular polytope-based approach, in this work, we explored different vaccine candidates based on selected, tandemly arranged cHPV-L2 epitopes fused to thioredoxin (Trx) as a scaffold protein. Upon conversion to heptameric nanoparticles with the use of a genetically fused oligomerization domain, our candidate Trx-L2 vaccines induce broadly neutralizing immune responses against 19 cHPV in guinea pigs. Similar findings were obtained in mice, where protection against virus challenge was also achieved via passive transfer of immune sera. Remarkably, immunization with the candidate cHPV vaccines also induced immune responses against several mucosal low- and high-risk HPV types, including HPV16 and 18. Based on cumulative immunogenicity data but also on ease and yield of production, we identified a lead vaccine candidate bearing 12 different cHPV-L2 epitopes that holds great promise as a scalable and GMP production-compatible lead molecule for the prevention of post-transplantation skin lesions caused by cHPV infection.

Enhanced immunogenicity of a positively supercharged archaeon thioredoxin scaffold as a cell-penetrating antigen carrier for peptide vaccines.

Polycationic resurfaced proteins hold great promise as cell-penetrating bioreagents but their use as carriers for the intracellular delivery of peptide immuno-epitopes has not thus far been explored. Here, we report on the construction and functional characterization of a positively supercharged derivative of Pyrococcus furiosus thioredoxin (PfTrx), a thermally hyperstable protein we have previously validated as a peptide epitope display and immunogenicity enhancing scaffold. Genetic conversion of 13 selected amino acids to lysine residues conferred to PfTrx a net charge of +21 (starting from the -1 charge of the wild-type protein), along with the ability to bind nucleic acids. In its unfused form, +21 PfTrx was readily internalized by HeLa cells and displayed a predominantly cytosolic localization. A different intracellular distribution was observed for a +21 PfTrx-eGFP fusion protein, which although still capable of cell penetration was predominantly localized within endosomes. A mixed cytosolic/endosomal partitioning was observed for a +21 PfTrx derivative harboring three tandemly repeated copies of a previously validated HPV16-L2 (aa 20-38) B-cell epitope grafted to the display site of thioredoxin. Compared to its wild-type counterpart, the positively supercharged antigen induced a faster immune response and displayed an overall superior immunogenicity, including a substantial degree of self-adjuvancy. Altogether, the present data point to +21 PfTrx as a promising novel carrier for intracellular antigen delivery and the construction of potentiated recombinant subunit vaccines.

A respirable HPV-L2 dry-powder vaccine with GLA as amphiphilic lubricant and immune-adjuvant.

Vaccines not requiring cold-chain storage/distribution and suitable for needle-free delivery are urgently needed. Pulmonary administration is one of the most promising non-parenteral routes for vaccine delivery. Through a multi-component excipient and spray-drying approach, we engineered highly respirable dry-powder vaccine particles containing a three-fold repeated peptide epitope derived from human papillomavirus (HPV16) minor capsid protein L2 displayed on Pyrococcus furious thioredoxin as antigen. A key feature of our engineering approach was the use of the amphiphilic endotoxin derivative glucopyranosyl lipid A (GLA) as both a coating agent enhancing particle de-aggregation and respirability as well as a built-in immune-adjuvant. Following an extensive characterization of the in vitro aerodynamic performance, lung deposition was verified in vivo by intratracheal administration in mice of a vaccine powder containing a fluorescently labeled derivative of the antigen. This was followed by a short-term immunization study that highlighted the ability of the GLA-adjuvanted vaccine powder to induce an anti-L2 systemic immune response comparable to (or even better than) that of the subcutaneously administered liquid-form vaccine. Despite the very short-term immunization conditions employed for this preliminary vaccination experiment, the intratracheally administered dry-powder, but not the subcutaneously injected liquid-state, vaccine induced consistent HPV neutralizing responses. Overall, the present data provide proof-of-concept validation of a new formulation design to produce a dry-powder vaccine that may be easily transferred to other antigens.

Degenerate CD8 Epitopes Mapping to Structurally Constrained Regions of the Spike Protein: A T Cell-Based Way-Out From the SARS-CoV-2 Variants Storm.

There is an urgent need for new generation anti-SARS-Cov-2 vaccines in order to increase the efficacy of immunization and its broadness of protection against viral variants that are continuously arising and spreading. The effect of variants on protective immunity afforded by vaccination has been mostly analyzed with regard to B cell responses. This analysis revealed variable levels of cross-neutralization capacity for presently available SARS-Cov-2 vaccines. Despite the dampened immune responses documented for some SARS-Cov-2 mutations, available vaccines appear to maintain an overall satisfactory protective activity against most variants of concern (VoC). This may be attributed, at least in part, to cell-mediated immunity. Indeed, the widely multi-specific nature of CD8 T cell responses should allow to avoid VoC-mediated viral escape, because mutational inactivation of a given CD8 T cell epitope is expected to be compensated by the persistent responses directed against unchanged co-existing CD8 epitopes. This is particularly relevant because some immunodominant CD8 T cell epitopes are located within highly conserved SARS-Cov-2 regions that cannot mutate without impairing SARS-Cov-2 functionality. Importantly, some of these conserved epitopes are degenerate, meaning that they are able to associate with different HLA class I molecules and to be simultaneously presented to CD8 T cell populations of different HLA restriction. Based on these concepts, vaccination strategies aimed at potentiating the stimulatory effect on SARS-Cov-2-specific CD8 T cells should greatly enhance the efficacy of immunization against SARS-Cov-2 variants. Our review recollects, discusses and puts into a translational perspective all available experimental data supporting these "hot" concepts, with special emphasis on the structural constraints that limit SARS-CoV-2 S-protein evolution and on potentially invariant and degenerate CD8 epitopes that lend themselves as excellent candidates for the rational development of next-generation, CD8 T-cell response-reinforced, COVID-19 vaccines.

Broad Neutralization Responses Against Oncogenic Human Papillomaviruses Induced by a Minor Capsid L2 Polytope Genetically Incorporated Into Bacterial Ferritin Nanoparticles.

Cervical cancer remains a global health burden despite the introduction of highly effective vaccines for the prophylaxis of causative human papillomavirus infection (HPV). Current efforts to eradicate cervical cancer focus on the development of broadly protective, cost-effective approaches. HPV minor capsid protein L2 is being recognized as a promising alternative to the major capsid protein L1 because of its ability to induce responses against a wider range of different HPV types. However, a major limitation of L2 as a source of cross-neutralizing epitopes is its lower immunogenicity compared to L1 when assembled into VLPs. Various approaches have been proposed to overcome this limitation, we developed and tested ferritin-based bio-nanoparticles displaying tandemly repeated L2 epitopes from eight different HPV types grafted onto the surface of Pyrococcus furiosus thioredoxin (Pf Trx). Genetic fusion of the Pf Trx-L2(8x) module to P. furiosus ferritin (Pf Fe) did not interfere with ferritin self-assembly into an octahedral structure composed by 24 protomers. In guinea pigs and mice, the ferritin super-scaffolded, L2 antigen induced a broadly neutralizing antibody response covering 14 oncogenic and two non-oncogenic HPV types. Immune-responsiveness lasted for at least one year and the resulting antibodies also conferred protection in a cervico-vaginal mouse model of HPV infection. Given the broad organism distribution of thioredoxin and ferritin, we also verified the lack of cross-reactivity of the antibodies elicited against the scaffolds with human thioredoxin or ferritin. Altogether, the results of this study point to P. furiosus ferritin nanoparticles as a robust platform for the construction of peptide-epitope-based HPV vaccines.

Combined prophylactic and therapeutic immune responses against human papillomaviruses induced by a thioredoxin-based L2-E7 nanoparticle vaccine.

Global burden of cervical cancer, the most common cause of mortality caused by human papillomavirus (HPV), is expected to increase during the next decade, mainly because current alternatives for HPV vaccination and cervical cancer screening programs are costly to be established in low-and-middle income countries. Recently, we described the development of the broadly protective, thermostable vaccine antigen Trx-8mer-OVX313 based on the insertion of eight different minor capsid protein L2 neutralization epitopes into a thioredoxin scaffold from the hyperthermophilic archaeon Pyrococcus furiosus and conversion of the resulting antigen into a nanoparticle format (median radius ~9 nm) upon fusion with the heptamerizing OVX313 module. Here we evaluated whether the engineered thioredoxin scaffold, in addition to humoral immune responses, can induce CD8+ T-cell responses upon incorporation of MHC-I-restricted epitopes. By systematically examining the contribution of individual antigen modules, we demonstrated that B-cell and T-cell epitopes can be combined into a single antigen construct without compromising either immunogenicity. While CD8+ T-cell epitopes had no influence on B-cell responses, the L2 polytope (8mer) and OVX313-mediated heptamerization of the final antigen significantly increased CD8+ T-cell responses. In a proof-of-concept experiment, we found that vaccinated mice remained tumor-free even after two consecutive tumor challenges, while unvaccinated mice developed tumors. A cost-effective, broadly protective vaccine with both prophylactic and therapeutic properties represents a promising option to overcome the challenges associated with prevention and treatment of HPV-caused diseases.

Mechanistic insights on the mode of action of an antiproliferative thiosemicarbazone-nickel complex revealed by an integrated chemogenomic profiling study.

Thiosemicarbazones (TSC) and their metal complexes display diverse biological activities and are active against multiple pathological conditions ranging from microbial infections to abnormal cell proliferation. Ribonucleotide reductase (RNR) is considered one of the main targets of TSCs, yet, the existence of additional targets, differently responsible for the multifaceted activities of TSCs and their metal complexes has been proposed. To set the basis for a more comprehensive delineation of their mode of action, we chemogenomically profiled the cellular effects of bis(citronellalthiosemicarbazonato)nickel(II) [Ni(S-tcitr)2] using the unicellular eukaryote Saccharomyces cerevisiae as a model organism. Two complementary genomic phenotyping screens led to the identification of 269 sensitive and 56 tolerant deletion mutant strains and of 14 genes that when overexpressed make yeast cells resistant to an otherwise lethal concentration of Ni(S-tcitr)2. Chromatin remodeling, cytoskeleton organization, mitochondrial function and iron metabolism were identified as lead cellular processes responsible for Ni(S-tcitr)2 toxicity. The latter process, and particularly glutaredoxin-mediated iron loading of RNR, was found to be affected by Ni(S-tcitr)2. Given the multiple pathways regulated by glutaredoxins, targeting of these proteins by Ni(S-tcitr)2 can negatively affect various core cellular processes that may critically contribute to Ni(S-tcitr)2 cytotoxicity.

Targeting p53 and histone methyltransferases restores exhausted CD8+ T cells in HCV infection.

Hepatitis C virus infection (HCV) represents a unique model to characterize, from early to late stages of infection, the T cell differentiation process leading to exhaustion of human CD8+ T cells. Here we show that in early HCV infection, exhaustion-committed virus-specific CD8+ T cells display a marked upregulation of transcription associated with impaired glycolytic and mitochondrial functions, that are linked to enhanced ataxia-telangiectasia mutated (ATM) and p53 signaling. After evolution to chronic infection, exhaustion of HCV-specific T cell responses is instead characterized by a broad gene downregulation associated with a wide metabolic and anti-viral function impairment, which can be rescued by histone methyltransferase inhibitors. These results have implications not only for treatment of HCV-positive patients not responding to last-generation antivirals, but also for other chronic pathologies associated with T cell dysfunction, including cancer.

Flavonoid-Derived Human Phenyl-γ-Valerolactone Metabolites Selectively Detoxify Amyloid-ß Oligomers and Prevent Memory Impairment in a Mouse Model of Alzheimer's Disease.

SCOPE: Amyloid-ß oligomers (AßO) are causally related to Alzheimer's disease (AD). Dietary natural compounds, especially flavonoids and flavan-3-ols, hold great promise as potential AD-preventive agents but their host and gut microbiota metabolism complicates identification of the most relevant bioactive species. This study aims to investigate the ability of a comprehensive set of phenyl-γ-valerolactones (PVL), the main circulating metabolites of flavan-3-ols and related dietary compounds in humans, to prevent AßO-mediated toxicity. METHODS AND RESULTS: The anti-AßO activity of PVLs is examined in different cell model systems using a highly toxic ß-oligomer-forming polypeptide (ß23) as target toxicant. Multiple PVLs, and particularly the monohydroxylated 5-(4'-hydroxyphenyl)-γ-valerolactone metabolite [(4'-OH)-PVL], relieve ß-oligomer-induced cytotoxicity in yeast and mammalian cells. As revealed by atomic force microscopy (AFM) and other in vitro assays, (4'-OH)-PVL interferes with AßO (but not fibril) assembly and actively remodels preformed AßOs into nontoxic amorphous aggregates. In keeping with the latter mode of action, treatment of AßOs with (4'-OH)-PVL prior to brain injection strongly reduces memory deterioration as well as neuroinflammation in a mouse model of AßO-induced memory impairment. CONCLUSION: PVLs, which have been validated as biomarkers of the dietary intake of flavan-3-ols, lend themselves as novel AßO-selective, candidate AD-preventing compounds.

New Antimicrobials Targeting Bacterial RNA Polymerase Holoenzyme Assembly Identified with an in Vivo BRET-Based Discovery Platform.

Bacterial resistance represents a major health threat worldwide, and the development of new therapeutics, including innovative antibiotics, is urgently needed. We describe a discovery platform, centered on in silico screening and in vivo bioluminescence resonance energy transfer in yeast cells, for the identification of new antimicrobials that, by targeting the protein-protein interaction between the ß'-subunit and the initiation factor σ70 of bacterial RNA polymerase, inhibit holoenzyme assembly and promoter-specific transcription. Out of 34 000 candidate compounds, we identified seven hits capable of interfering with this interaction. Two derivatives of one of these hits proved to be effective in inhibiting transcription in vitro and growth of the Gram-positive pathogens Staphylococcus aureus and Listeria monocytogenes. Upon supplementation of a permeability adjuvant, one derivative also effectively inhibited Escherichia coli growth. On the basis of the chemical structures of these inhibitors, we generated a ligand-based pharmacophore model that will guide the rational discovery of increasingly effective antibacterial agents.

Pezizomycetes genomes reveal the molecular basis of ectomycorrhizal truffle lifestyle.

Tuberaceae is one of the most diverse lineages of symbiotic truffle-forming fungi. To understand the molecular underpinning of the ectomycorrhizal truffle lifestyle, we compared the genomes of Piedmont white truffle (Tuber magnatum), Périgord black truffle (Tuber melanosporum), Burgundy truffle (Tuber aestivum), pig truffle (Choiromyces venosus) and desert truffle (Terfezia boudieri) to saprotrophic Pezizomycetes. Reconstructed gene duplication/loss histories along a time-calibrated phylogeny of Ascomycetes revealed that Tuberaceae-specific traits may be related to a higher gene diversification rate. Genomic features in Tuber species appear to be very similar, with high transposon content, few genes coding lignocellulose-degrading enzymes, a substantial set of lineage-specific fruiting-body-upregulated genes and high expression of genes involved in volatile organic compound metabolism. Developmental and metabolic pathways expressed in ectomycorrhizae and fruiting bodies of T. magnatum and T. melanosporum are unexpectedly very similar, owing to the fact that they diverged ~100 Ma. Volatile organic compounds from pungent truffle odours are not the products of Tuber-specific gene innovations, but rely on the differential expression of an existing gene repertoire. These genomic resources will help to address fundamental questions in the evolution of the truffle lifestyle and the ecology of fungi that have been praised as food delicacies for centuries.

Author Correction: A family of archaea-like carboxylesterases preferentially expressed in the symbiotic phase of the mycorrhizal fungus Tuber melanosporum.

A correction to this article has been published and is linked from the HTML and the PDF versions of this paper. The error has been fixed in the paper.

Doxycycline counteracts neuroinflammation restoring memory in Alzheimer's disease mouse models.

ß-Amyloid oligomers (AßOs) and neuroinflammation are 2 main culprits to counteract in Alzheimer's disease (AD). Doxycycline (DOXY) is a second generation antibiotic of the tetracycline class that are promising drugs tested in many clinical trials for a number of different pathologies. DOXY is endowed with antiamyloidogenic properties and better crosses the blood-brain barrier, but its efficacy has never been tested in AD mice. We herein show that 15- to 16-month-old APP/PS1dE9 (APP/PS1) AD mice receiving DOXY under different treatment regimens recovered their memory without plaque reduction. An acute DOXY treatment was, also, sufficient to improve APP/PS1 mouse memory, suggesting an action against soluble AßOs. This was confirmed in an AßO-induced mouse model, where the AßO-mediated memory impairment was abolished by a DOXY pretreatment. Although AßOs induce memory impairment through glial activation, assessing the anti-inflammatory action of DOXY, we found that in both the AßO-treated and APP/PS1 mice, the memory recovery was associated with a lower neuroinflammation. Our data promote DOXY as a hopeful repositioned drug counteracting crucial neuropathological AD targets.