Publications by authors named "Simone Ottonello"

77 Publications

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

Front Immunol 2020 4;11:606569. Epub 2020 Dec 4.

Research Group Tumorvirus-Specific Vaccination Strategies, Research Program Infection Inflammation & Cancer, German Cancer Research Center, Heidelberg, Germany.

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 thioredoxin (Pf Trx). Genetic fusion of the Pf Trx-L2(8x) module to 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 ferritin nanoparticles as a robust platform for the construction of peptide-epitope-based HPV vaccines.
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http://dx.doi.org/10.3389/fimmu.2020.606569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7746619PMC
December 2020

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

PLoS Pathog 2020 09 4;16(9):e1008827. Epub 2020 Sep 4.

German Cancer Research Center, Heidelberg, Germany.

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.
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http://dx.doi.org/10.1371/journal.ppat.1008827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7498061PMC
September 2020

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

Sci Rep 2020 06 29;10(1):10524. Epub 2020 Jun 29.

Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area Delle Scienze, 11/A, 43124, Parma, Italy.

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)] 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). Chromatin remodeling, cytoskeleton organization, mitochondrial function and iron metabolism were identified as lead cellular processes responsible for Ni(S-tcitr) toxicity. The latter process, and particularly glutaredoxin-mediated iron loading of RNR, was found to be affected by Ni(S-tcitr). Given the multiple pathways regulated by glutaredoxins, targeting of these proteins by Ni(S-tcitr) can negatively affect various core cellular processes that may critically contribute to Ni(S-tcitr) cytotoxicity.
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http://dx.doi.org/10.1038/s41598-020-67439-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324377PMC
June 2020

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

Nat Commun 2020 01 30;11(1):604. Epub 2020 Jan 30.

Department of Medicine and Surgery, University of Parma, Parma, Italy.

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.
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http://dx.doi.org/10.1038/s41467-019-14137-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992697PMC
January 2020

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

Mol Nutr Food Res 2020 03 16;64(5):e1900890. Epub 2020 Jan 16.

Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, 43124, Parma, Italy.

Scope: Amyloid-β oligomers (AβO) 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 AβO-mediated toxicity.

Methods And Results: The anti-AβO activity of PVLs is examined in different cell model systems using a highly toxic β-oligomer-forming polypeptide (β23) as target toxicant. Multiple PVLs, and particularly the monohydroxylated 5-(4'-hydroxyphenyl)-γ-valerolactone metabolite [(4'-OH)-PVL], relieve β-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 AβO (but not fibril) assembly and actively remodels preformed AβOs into nontoxic amorphous aggregates. In keeping with the latter mode of action, treatment of AβOs with (4'-OH)-PVL prior to brain injection strongly reduces memory deterioration as well as neuroinflammation in a mouse model of AβO-induced memory impairment.

Conclusion: PVLs, which have been validated as biomarkers of the dietary intake of flavan-3-ols, lend themselves as novel AβO-selective, candidate AD-preventing compounds.
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http://dx.doi.org/10.1002/mnfr.201900890DOI Listing
March 2020

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

ACS Chem Biol 2019 08 30;14(8):1727-1736. Epub 2019 Jul 30.

Laboratory of Biochemistry and Molecular Biology, Department of Chemistry, Life Sciences and Environmental Sustainability , University of Parma , 43124 Parma , Italy.

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 screening and bioluminescence resonance energy transfer in yeast cells, for the identification of new antimicrobials that, by targeting the protein-protein interaction between the β'-subunit and the initiation factor σ 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 and growth of the Gram-positive pathogens and . Upon supplementation of a permeability adjuvant, one derivative also effectively inhibited 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.
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http://dx.doi.org/10.1021/acschembio.9b00178DOI Listing
August 2019

Pezizomycetes genomes reveal the molecular basis of ectomycorrhizal truffle lifestyle.

Nat Ecol Evol 2018 12 12;2(12):1956-1965. Epub 2018 Nov 12.

CNR-IBBR, Istituto di Bioscienze e Biorisorse, UOS di Perugia, Perugia, Italy.

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.
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http://dx.doi.org/10.1038/s41559-018-0710-4DOI Listing
December 2018

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

Sci Rep 2018 Aug 29;8(1):13173. Epub 2018 Aug 29.

Department of Chemical Life Sciences and Environmental Sustainability, University of Parma, Parco Area delle Scienze 23/A, 43124, Parma, Italy.

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.
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http://dx.doi.org/10.1038/s41598-018-29606-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115439PMC
August 2018

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

Neurobiol Aging 2018 10 12;70:128-139. Epub 2018 Jun 12.

Department of Neuroscience, IRCCS, Istituto di Ricerhe Farmacologiche Mario Negri, Milano, Italy. Electronic address:

β-Amyloid oligomers (AβOs) 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 AβOs. This was confirmed in an AβO-induced mouse model, where the AβO-mediated memory impairment was abolished by a DOXY pretreatment. Although AβOs induce memory impairment through glial activation, assessing the anti-inflammatory action of DOXY, we found that in both the AβO-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.
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http://dx.doi.org/10.1016/j.neurobiolaging.2018.06.002DOI Listing
October 2018

Broadly neutralizing antiviral responses induced by a single-molecule HPV vaccine based on thermostable thioredoxin-L2 multiepitope nanoparticles.

Sci Rep 2017 12 21;7(1):18000. Epub 2017 Dec 21.

Department of Chemical Life Sciences & Environmental Sustainability, University of Parma, Parma, Italy.

Vaccines targeting the human papillomavirus (HPV) minor capsid protein L2 are emerging as chemico-physically robust and broadly protective alternatives to the current HPV (L1-VLP) vaccines. We have previously developed a trivalent L2 vaccine prototype exploiting Pyrococcus furiosus thioredoxin (PfTrx) as a thermostable scaffold for the separate presentation of three distinct HPV L2(20-38) epitopes. With the aim of achieving a highly immunogenic, yet simpler and more GMP-production affordable formulation, we report here on a novel thermostable nanoparticle vaccine relying on genetic fusion of PfTrx-L2 with the heptamerizing coiled-coil polypeptide OVX313. A prototype HPV16 monoepitope version of this nanoparticle vaccine (PfTrx-L2-OVX313; median radius: 8.6 ± 1.0 nm) proved to be approximately 10-fold more immunogenic and with a strikingly enhanced cross-neutralization capacity compared to its monomeric counterpart. Vaccine-induced (cross-)neutralizing responses were further potentiated in a multiepitope derivative displaying eight different L2(20-38) epitopes, which elicited neutralizing antibodies against 10 different HPVs including three viral types not represented in the vaccine. Considering the prospective safety of the PfTrx scaffold and of the OVX313 heptamerization module, PfTrx-OVX313 nanoparticles lend themselves as robust L2-based immunogens with a high translational potential as a 3 generation HPV vaccine, but also as a novel and extremely versatile peptide-antigen presentation platform.
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http://dx.doi.org/10.1038/s41598-017-18177-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5740060PMC
December 2017

Minor Capsid Protein L2 Polytope Induces Broad Protection against Oncogenic and Mucosal Human Papillomaviruses.

J Virol 2018 02 30;92(4). Epub 2018 Jan 30.

German Cancer Research Center, Heidelberg, Germany

The amino terminus of the human papillomavirus (HPV) minor capsid protein L2 contains a major cross-neutralization epitope which provides the basis for the development of a broadly protecting HPV vaccine. A wide range of protection against different HPV types would eliminate one of the major drawbacks of the commercial, L1-based prophylactic vaccines. Previously, we have reported that insertion of the L2 epitope into a scaffold composed of bacterial thioredoxin protein generates a potent antigen inducing comprehensive protection against different animal and human papillomaviruses. We also reported, however, that although protection is broad, some oncogenic HPV types escape the neutralizing antibody response, if L2 epitopes from single HPV types are used as immunogen. We were able to compensate for this by applying a mix of thioredoxin proteins carrying L2 epitopes from HPV16, -31, and -51. As the development of a cost-efficient HPV prophylactic vaccines is one of our objectives, this approach is not feasible as it requires the development of multiple good manufacturing production processes in combination with a complex vaccine formulation. Here, we report the development of a thermostable thioredoxin-based single-peptide vaccine carrying an L2 polytope of up to 11 different HPV types. The L2 polytope antigens have excellent abilities in respect to broadness of protection and robustness of induced immune responses. To further increase immunogenicity, we fused the thioredoxin L2 polytope antigen with a heptamerization domain. In the final vaccine design, we achieve protective responses against all 14 oncogenic HPV types that we have analyzed plus the low-risk HPVs 6 and 11 and a number of cutaneous HPVs. Infections by a large number of human papillomaviruses lead to malignant and nonmalignant disease. Current commercial vaccines based on virus-like particles (VLPs) effectively protect against some HPV types but fail to do so for most others. Further, only about a third of all countries have access to the VLP vaccines. The minor capsid protein L2 has been shown to contain so-called neutralization epitopes within its N terminus. We designed polytopes comprising the L2 epitope amino acids 20 to 38 of up to 11 different mucosal HPV types and inserted them into the scaffold of thioredoxin derived from a thermophile archaebacterium. The antigen induced neutralizing antibody responses in mice and guinea pigs against 26 mucosal and cutaneous HPV types. Further, addition of a heptamerization domain significantly increased the immunogenicity. The final vaccine design comprising a heptamerized L2 8-mer thioredoxin single-peptide antigen with excellent thermal stability might overcome some of the limitations of the current VLP vaccines.
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http://dx.doi.org/10.1128/JVI.01930-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790957PMC
February 2018

Induction of Antihuman C-C Chemokine Receptor Type 5 Antibodies by a Bovine Herpesvirus Type-4 Based Vector.

Front Immunol 2017 25;8:1402. Epub 2017 Oct 25.

Department of Medical Veterinary Science, University of Parma, Parma, Italy.

Bovine herpesvirus 4 (BoHV-4) is a promising vector for the delivery and intracellular expression of recombinant antigens and can thus be considered as a new prototype vaccine formulation system. An interesting, and actively pursued, antigen in the context of human immunodeficiency virus (HIV) infection prophylaxis (and therapy) is the C-C chemokine receptor type 5 (CCR5) co-receptor, whose blockage by specific antibodies has been shown to inhibit both viral entry and cell-to-cell transmission of the virus. Building on our previous work on the BoHV-4 vector system, we have engineered and tested a replication-competent derivative of BoHV-4 (BoHV-4-CMV-hCCR5ΔTK) bearing a human CCR5 (hCCR5) expression cassette. We show here that CCR5 is indeed expressed at high levels in multiple types of BoHV-4-CMV-hCCR5ΔTK-infected cells. More importantly, two intravenous inoculations of CCR5-expressing BoHV-4 virions into rabbits led to the production of anti-CCR5 antibodies capable of reacting with the CCR5 receptor exposed on the surface of HEK293T cells through specific recognition of the amino-terminal region (aa 14-34) of the protein. Given the growing interest for anti-CCR5 immunization as an HIV control strategy and the many advantages of virus-based immunogen formulations (especially for poorly immunogenic or self-antigens), the results reported in this study provide preliminary validation of BoHV-4 as a safe viral vector suitable for CCR5 vaccination.
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http://dx.doi.org/10.3389/fimmu.2017.01402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5660961PMC
October 2017

A family of archaea-like carboxylesterases preferentially expressed in the symbiotic phase of the mychorrizal fungus Tuber melanosporum.

Sci Rep 2017 08 9;7(1):7628. Epub 2017 Aug 9.

Department of Chemical Life Sciences and Environmental Sustainability, University of Parma, Parco Area delle Scienze 23/A, 43124, Parma, Italy.

An increasing number of esterases is being revealed by (meta) genomic sequencing projects, but few of them are functionally/structurally characterized, especially enzymes of fungal origin. Starting from a three-member gene family of secreted putative "lipases/esterases" preferentially expressed in the symbiotic phase of the mycorrhizal fungus Tuber melanosporum ("black truffle"), we show here that these enzymes (TmelEST1-3) are dimeric, heat-resistant carboxylesterases capable of hydrolyzing various short/medium chain p-nitrophenyl esters. TmelEST2 was the most active (kcat = 2302 s for p-nitrophenyl-butyrate) and thermally stable (T = 68.3 °C), while TmelEST3 was the only one displaying some activity on tertiary alcohol esters. X-ray diffraction analysis of TmelEST2 revealed a classical α/β hydrolase-fold structure, with a network of dimer-stabilizing intermolecular interactions typical of archaea esterases. The predicted structures of TmelEST1 and 3 are overall quite similar to that of TmelEST2 but with some important differences. Most notably, the much smaller volume of the substrate-binding pocket and the more acidic electrostatic surface profile of TmelEST1. This was also the only TmelEST capable of hydrolyzing feruloyl-esters, suggestinng a possible role in root cell-wall deconstruction during symbiosis establishment. In addition to their potential biotechnological interest, TmelESTs raise important questions regarding the evolutionary recruitment of archaea-like enzymes into mesophilic subterranean fungi such as truffles.
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http://dx.doi.org/10.1038/s41598-017-08007-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5550427PMC
August 2017

Photoreceptors in the dark: A functional white collar-like complex and other putative light-sensing components encoded by the genome of the subterranean fungus Tuber melanosporum.

Fungal Biol 2017 03 5;121(3):253-263. Epub 2017 Jan 5.

Pasteur Cenci Bolognetti Foundation c/o Department of Biology and Biotechnology "Charles Darwin", Sapienza University, 00185 Rome, Italy; Institute of Biology and Molecular Pathology, CNR, 00185 Roma, Italy. Electronic address:

Light is perceived and transduced by fungi, where it modulates processes as diverse as growth and morphogenesis, sexual development and secondary metabolism. A special case in point is that of fungi with a subterranean, light-shielded habitat such as Tuber spp. Using as reference the genome sequence of the black truffle Tuber melanosporum, we used bioinformatic prediction tools and expression data to gain insight on the photoreceptor systems of this hypogeous ectomycorrhizal fungus. These include a chromophore-less opsin, a putative red-light-sensing phytochrome not expressed at detectable levels in any of the examined lifecycle stages, and a nearly canonical two-component (WC-1/WC-2) photoreceptor system similar to the Neurospora white collar complex (WCC). Multiple evidence, including expression at relatively high levels in all lifecycle stages except for fruiting-bodies and the results of heterologous functional complementation experiments conducted in Neurospora, suggests that the Tuber WCC is likely functional and capable of responding to blue-light. The other putative T. melanosporum photoreceptor components, especially the chromophore-less opsin and the likely non-functional phytochrome, may instead represent signatures of adaptation to a hypogeous (light-shielded) lifestyle.
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http://dx.doi.org/10.1016/j.funbio.2016.12.003DOI Listing
March 2017

Targeting mitochondrial dysfunction can restore antiviral activity of exhausted HBV-specific CD8 T cells in chronic hepatitis B.

Nat Med 2017 Mar 6;23(3):327-336. Epub 2017 Feb 6.

Laboratory of Viral Immunopathology, Unit of Infectious Diseases and Hepatology, Azienda Ospedaliero-Universitaria of Parma, Parma, Italy.

Hepatitis B virus (HBV)-specific CD8 T cells are functionally exhausted in chronic hepatitis B infection, and this condition can be corrected only partially through the modulation of inhibitory pathways, which suggests that a more complex molecular interplay underlies T cell exhaustion. To gain broader insight into this process and identify additional targets for the restoration of T cell function, we compared the transcriptome profiles of HBV-specific CD8 T cells from patients with acute and chronic disease with those of HBV-specific CD8 T cells from patients able to resolve HBV infection spontaneously and influenza (FLU)-specific CD8 T cells from healthy participants. The results indicate that exhausted HBV-specific CD8 T cells are markedly impaired at multiple levels and show substantial downregulation of various cellular processes centered on extensive mitochondrial alterations. A notable improvement of mitochondrial and antiviral CD8 functions was elicited by mitochondrion-targeted antioxidants, which suggests a central role for reactive oxygen species (ROS) in T cell exhaustion. Thus, mitochondria represent promising targets for novel reconstitution therapies to treat chronic hepatitis B infection.
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http://dx.doi.org/10.1038/nm.4275DOI Listing
March 2017

Dry and wet approaches for genome-wide functional annotation of conventional and unconventional transcriptional activators.

Comput Struct Biotechnol J 2016 29;14:262-70. Epub 2016 Jun 29.

Biochemistry and Molecular Biology Unit, Laboratory of Functional Genomics and Protein Engineering, Department of Life Sciences, University of Parma, Parco Area delle Scienze 23/A, 43124 Parma, Italy.

Transcription factors (TFs) are master gene products that regulate gene expression in response to a variety of stimuli. They interact with DNA in a sequence-specific manner using a variety of DNA-binding domain (DBD) modules. This allows to properly position their second domain, called "effector domain", to directly or indirectly recruit positively or negatively acting co-regulators including chromatin modifiers, thus modulating preinitiation complex formation as well as transcription elongation. At variance with the DBDs, which are comprised of well-defined and easily recognizable DNA binding motifs, effector domains are usually much less conserved and thus considerably more difficult to predict. Also not so easy to identify are the DNA-binding sites of TFs, especially on a genome-wide basis and in the case of overlapping binding regions. Another emerging issue, with many potential regulatory implications, is that of so-called "moonlighting" transcription factors, i.e., proteins with an annotated function unrelated to transcription and lacking any recognizable DBD or effector domain, that play a role in gene regulation as their second job. Starting from bioinformatic and experimental high-throughput tools for an unbiased, genome-wide identification and functional characterization of TFs (especially transcriptional activators), we describe both established (and usually well affordable) as well as newly developed platforms for DNA-binding site identification. Selected combinations of these search tools, some of which rely on next-generation sequencing approaches, allow delineating the entire repertoire of TFs and unconventional regulators encoded by the any sequenced genome.
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http://dx.doi.org/10.1016/j.csbj.2016.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941109PMC
July 2016

Virus-Mediated Metalloproteinase 1 Induction Revealed by Transcriptome Profiling of Bovine Herpesvirus 4-Infected Bovine Endometrial Stromal Cells.

Biol Reprod 2016 07 8;95(1):12. Epub 2016 Jun 8.

Department of Medical-Veterinary Science, University of Parma, Parma, Italy

Viral infections can cause genital tract disorders (including abortion) in cows, and bovine herpesvirus 4 (BoHV-4) is often present in endometritis-affected animals. A major problem with cattle uterine viral infections in general, and BoHV-4 in particular, is our limited understanding of the pathogenic role(s) that these infections play in the endometrium. A similar lack of knowledge holds for the molecular mechanisms utilized, and the host cell pathways affected, by BoHV-4. To begin to fill these gaps, we set up optimized conditions for BoHV-4 infection of a pure population of bovine endometrial stromal cells (BESCs) to be used as source material for RNA sequencing-based transcriptome profiling. Many genes were found to be upregulated (417) or downregulated (181) after BoHV-4 infection. As revealed by enrichment functional analysis on differentially expressed genes, BoHV-4 infection affects various pathways related to cell proliferation and cell surface integrity, at least three of which were centered on upregulation of matrix metalloproteinase 1 (MMP1) and interleukin 8 (IL8). This was confirmed by reverse transcription PCR, real-time PCR, Western-immunoblot analysis, and a luciferase assay with a bovine MMP1-specific promoter reporter construct. Further, it was found that MMP1 transcription was upregulated by the BoHV-4 transactivator IE2/RTA, leading to abnormally high metalloproteinase tissue levels, potentially leading to defective endometrium healing and unresolved inflammation. Based on these findings, we propose a new model for BoHV-4 action centered on IE2-mediated MMP1 upregulation and novel therapeutic interventions based on IFN gamma-mediated MMP1 downregulation.
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http://dx.doi.org/10.1095/biolreprod.116.139097DOI Listing
July 2016

Bovine herpesvirus 4-based vector delivering a hybrid rat/human HER-2 oncoantigen efficiently protects mice from autochthonous Her-2 mammary cancer.

Oncoimmunology 2016 Mar 31;5(3):e1082705. Epub 2015 Aug 31.

Department of Medical-Veterinary Science, University of Parma , Parma, Italy.

The epidermal growth factor receptor 2 (HER-2) oncogene is a major target for the immunotherapy of breast cancer. Following up to the therapeutic success achieved with Her-2-targeting monoclonal antibodies, immune-prophylactic approaches directed against Her-2 have also been investigated taking into account, and trying to overcome, Her-2 self-tolerance. Perhaps due to safety (and efficacy) concerns, the least explored anti-Her-2 active immunization strategy so far has been the one relying on viral-vectored vaccine formulations. Taking advantage of the favorable properties of bovine herpesvirus 4 (BoHV-4) in terms of safety and ease of manipulation as well as its previously documented ability to transduce and confer immunogenicity to heterologous antigens, we tested the ability of different recombinant HER-2-BoHV-4 immunogens to 8break tolerance and elicit a protective, anti-mammary tumor antibody response in HER-2 transgenic BALB-neuT mice. All the tested constructs expressed the HER-2 transgenes at high levels and elicited significant cellular immune responses in BALB/c mice upon administration via either DNA vaccination or viral infection. In BALB-neuT mice, instead, only the viral construct expressing the membrane-bound chimeric form of Her-2 protein (BoHV-4-RHuT-gD) elicited a humoral immune response that was more intense and earlier-appearing than that induced by DNA vaccination. In keeping with this observation, two administrations of BoHV-4-RHuT-gD effectively protected BALB-neuT mice from tumor formation, with 50% of vaccinated animals tumor-free after 30 weeks from immunization compared to 100% of animals exhibiting at least one palpable tumor in the case of animals vaccinated with the other BoHV-4-HER-2 constructs.
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http://dx.doi.org/10.1080/2162402X.2015.1082705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4839386PMC
March 2016

Secretory production of designed multipeptides displayed on a thermostable bacterial thioredoxin scaffold in Pichia pastoris.

Protein Expr Purif 2017 01 29;129:150-157. Epub 2016 Apr 29.

Department of Life Sciences, Biochemistry and Molecular Biology Unit, University of Parma, Italy. Electronic address:

Internal grafting of designed peptides to scaffold proteins is a valuable strategy for a variety of applications including recombinant peptide antigen construction. A peptide epitope from human papillomavirus (HPV) minor capsid protein L2 displayed on thioredoxin (Trx) has been validated preclinically as a broadly protective and low-cost alternative HPV vaccine. Focusing on thioredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus (PfTrx) as a scaffold, we have constructed a modified Pichia pastoris expression vector and used a PfTrx fusion derivative containing three tandemly repeated copies of a 19 amino acids peptide epitope from HPV-L2 for expression optimization and biochemical-immunological characterization of the Pichia-produced PfTrx-L2 antigen. We show that PfTrx-L2 is produced at high levels (up to 100 mg from a 100 ml starting culture using a multi-cycle induction protocol) and secreted into the culture medium as a highly enriched (>70% pure), non-glycosylated polypeptide that can be purified to homogeneity in a single step. Oxidation and aggregation state, thermal stability and immunogenicity of the endotoxin-free PfTrx-L2 antigen produced in P. pastoris were tested and found to be identical to those of the same antigen produced in Escherichia coli. Secretory production of endotoxin-free PfTrx-peptides in P. pastoris represents a cost- and time-effective alternative to E. coli production. Specifically designed for peptide antigens, the PfTrx-expression vector and conditions described herein are easily transferable to a variety of applications centred on the use of structurally constrained bioactive peptides as immune as well as target-specific binder reagents.
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http://dx.doi.org/10.1016/j.pep.2016.04.012DOI Listing
January 2017

Moonlighting transcriptional activation function of a fungal sulfur metabolism enzyme.

Sci Rep 2016 04 28;6:25165. Epub 2016 Apr 28.

Biochemistry and Molecular Biology Unit, Laboratory of Functional Genomics and Protein Engineering, Department of Life Sciences, University of Parma, Parco Area delle Scienze 23/A, 43124 Parma, Italy.

Moonlighting proteins, including metabolic enzymes acting as transcription factors (TF), are present in a variety of organisms but have not been described in higher fungi so far. In a previous genome-wide analysis of the TF repertoire of the plant-symbiotic fungus Tuber melanosporum, we identified various enzymes, including the sulfur-assimilation enzyme phosphoadenosine-phosphosulfate reductase (PAPS-red), as potential transcriptional activators. A functional analysis performed in the yeast Saccharomyces cerevisiae, now demonstrates that a specific variant of this enzyme, PAPS-red A, localizes to the nucleus and is capable of transcriptional activation. TF moonlighting, which is not present in the other enzyme variant (PAPS-red B) encoded by the T. melanosporum genome, relies on a transplantable C-terminal polypeptide containing an alternating hydrophobic/hydrophilic amino acid motif. A similar moonlighting activity was demonstrated for six additional proteins, suggesting that multitasking is a relatively frequent event. PAPS-red A is sulfur-state-responsive and highly expressed, especially in fruitbodies, and likely acts as a recruiter of transcription components involved in S-metabolism gene network activation. PAPS-red B, instead, is expressed at low levels and localizes to a highly methylated and silenced region of the genome, hinting at an evolutionary mechanism based on gene duplication, followed by epigenetic silencing of this non-moonlighting gene variant.
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http://dx.doi.org/10.1038/srep25165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848566PMC
April 2016

Thioredoxin-Displayed Multipeptide Immunogens.

Methods Mol Biol 2015 ;1348:137-51

Biochemistry and Molecular Biology Unit, Department of Life Sciences, University of Parma, Parma, Italy.

Fusion to carrier proteins is an effective strategy for stabilizing and providing immunogenicity to peptide epitopes. This is commonly achieved by cross-linking of chemically synthesized peptides to carrier proteins. An alternative approach is internal grafting of selected peptide epitopes to a scaffold protein via double stranded-oligonucleotide insertion or gene synthesis, followed by recombinant expression of the resulting chimeric polypeptide. The scaffold protein should confer immunogenicity to the stabilized and structurally constrained peptide, but also afford easy production of the antigen in recombinant form. A macromolecular scaffold that meets the above criteria is the redox protein thioredoxin, especially bacterial thioredoxin. Here we describe our current methodology for internal grafting of selected peptide epitopes to thioredoxin as tandemly arranged multipeptide repeats ("Thioredoxin Displayed Multipeptide Immunogens"), bacterial expression and purification of the recombinant thioredoxin-multipeptide fusion proteins and their use as antigens for the production of anti-peptide antibodies for prophylactic vaccine as well as diagnostic purposes.
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http://dx.doi.org/10.1007/978-1-4939-2999-3_14DOI Listing
June 2016

Elongator-dependent modification of cytoplasmic tRNALysUUU is required for mitochondrial function under stress conditions.

Nucleic Acids Res 2015 Sep 3;43(17):8368-80. Epub 2015 Aug 3.

Department of Life Sciences, University of Parma, 43124 Parma, Italy

To gain a wider view of the pathways that regulate mitochondrial function, we combined the effect of heat stress on respiratory capacity with the discovery potential of a genome-wide screen in Saccharomyces cerevisiae. We identified 105 new genes whose deletion impairs respiratory growth at 37°C by interfering with processes such as transcriptional regulation, ubiquitination and cytosolic tRNA wobble uridine modification via 5-methoxycarbonylmethyl-2-thiouridine formation. The latter process, specifically required for efficient decoding of AA-ending codons under stress conditions, was covered by multiple genes belonging to the Elongator (e.g. ELP3) and urmylation (e.g., NCS6) pathways. ELP3 or NCS6 deletants had impaired mitochondrial protein synthesis. Their respiratory deficiency was selectively rescued by overexpression of tRNA(Lys) UUU as well by overexpression of genes (BCK1 and HFM1) with a strong bias for the AAA codon read by this tRNA. These data extend the mitochondrial regulome, demonstrate that heat stress can impair respiration by disturbing cytoplasmic translation of proteins critically involved in mitochondrial function and document, for the first time, the involvement in such process of the Elongator and urmylation pathways. Given the conservation of these pathways, the present findings may pave the way to a better understanding of the human mitochondrial regulome in health and disease.
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http://dx.doi.org/10.1093/nar/gkv765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4787798PMC
September 2015

Robust In Vitro and In Vivo Neutralization against Multiple High-Risk HPV Types Induced by a Thermostable Thioredoxin-L2 Vaccine.

Cancer Prev Res (Phila) 2015 Oct 13;8(10):932-41. Epub 2015 Jul 13.

German Cancer Research Center, Heidelberg, Germany.

Current prophylactic virus-like particle (VLP) human papillomavirus (HPV) vaccines are based on the L1 major capsid protein and provide robust but virus type-restricted protection. Moreover, VLP vaccines have a high production cost, require cold-chain storage, and are thus not readily implementable in developing countries, which endure 85% of the cervical cancer-related death burden worldwide. In contrast with L1, immunization with minor capsid protein L2 elicits broad cross-neutralization, and we previously showed that insertion of a peptide spanning amino acids 20-38 of L2 into bacterial thioredoxin (Trx) greatly enhances its immunogenicity. Building on this finding, we use, here, four different neutralization assays to demonstrate that low doses of a trivalent Trx-L2 vaccine, incorporating L2(20-38) epitopes from HPV16, HPV31 and HPV51, and formulated in a human-compatible adjuvant, induce broadly protective responses. Specifically, we show that this vaccine, which uses a far-divergent archaebacterial thioredoxin as scaffold and is amenable to an easy one-step thermal purification, induces robust cross-neutralization against 12 of the 13 known oncogenic HPV types. Immune performance measured with two different in vitro neutralization assays was corroborated by the results of mouse cervico-vaginal challenge and passive transfer experiments indicating robust cross-protection also in vivo. Altogether, our results attest to the potential of Trx-L2 as a thermostable second-generation HPV vaccine particularly well suited for low-resource countries.
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http://dx.doi.org/10.1158/1940-6207.CAPR-15-0164DOI Listing
October 2015

In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse.

Elife 2015 Apr 7;4:e06205. Epub 2015 Apr 7.

Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, United States.

Methylation of cytosines (5(me)C) is a widespread heritable DNA modification. During mammalian development, two global demethylation events are followed by waves of de novo DNA methylation. In vivo mechanisms of DNA methylation establishment are largely uncharacterized. Here, we use Saccharomyces cerevisiae as a system lacking DNA methylation to define the chromatin features influencing the activity of the murine DNMT3B. Our data demonstrate that DNMT3B and H3K4 methylation are mutually exclusive and that DNMT3B is co-localized with H3K36 methylated regions. In support of this observation, DNA methylation analysis in yeast strains without Set1 and Set2 shows an increase of relative 5(me)C levels at the transcription start site and a decrease in the gene-body, respectively. We extend our observation to the murine male germline, where H3K4me3 is strongly anti-correlated while H3K36me3 correlates with accelerated DNA methylation. These results show the importance of H3K36 methylation for gene-body DNA methylation in vivo.
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http://dx.doi.org/10.7554/eLife.06205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4412109PMC
April 2015

A comprehensive resource of genomic, epigenomic and transcriptomic sequencing data for the black truffle Tuber melanosporum.

Gigascience 2014 30;3:25. Epub 2014 Oct 30.

Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, CA 90095, USA.

Background: Tuber melanosporum, also known in the gastronomic community as "truffle", features one of the largest fungal genomes (125 Mb) with an exceptionally high transposable element (TE) and repetitive DNA content (>58%). The main purpose of DNA methylation in fungi is TE silencing. As obligate outcrossing organisms, truffles are bound to a sexual mode of propagation, which together with TEs is thought to represent a major force driving the evolution of DNA methylation. Thus, it was of interest to examine if and how T. melanosporum exploits DNA methylation to maintain genome integrity.

Findings: We performed whole-genome DNA bisulfite sequencing and mRNA sequencing on different developmental stages of T. melanosporum; namely, fruitbody ("truffle"), free-living mycelium and ectomycorrhiza. The data revealed a high rate of cytosine methylation (>44%), selectively targeting TEs rather than genes with a strong preference for CpG sites. Whole genome DNA sequencing uncovered multiple TE-enriched, copy number variant regions bearing a significant fraction of hypomethylated and expressed TEs, almost exclusively in free-living mycelium propagated in vitro. Treatment of mycelia with 5-azacytidine partially reduced DNA methylation and increased TE transcription. Our transcriptome assembly also resulted in the identification of a set of novel transcripts from 614 genes.

Conclusions: The datasets presented here provide valuable and comprehensive (epi)genomic information that can be of interest for evolutionary genomics studies of multicellular (filamentous) fungi, in particular Ascomycetes belonging to the subphylum, Pezizomycotina. Evidence derived from comparative methylome and transcriptome analyses indicates that a non-exhaustive and partly reversible methylation process operates in truffles.
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http://dx.doi.org/10.1186/2047-217X-3-25DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4228822PMC
November 2014

Non-exhaustive DNA methylation-mediated transposon silencing in the black truffle genome, a complex fungal genome with massive repeat element content.

Genome Biol 2014 Jul 31;15(7):411. Epub 2014 Jul 31.

Background: We investigated how an extremely transposon element (TE)-rich organism such as the plant-symbiotic ascomycete truffle Tuber melanosporum exploits DNA methylation to cope with the more than 45,000 repeated elements that populate its genome.

Results: Whole-genome bisulfite sequencing performed on different developmental stages reveals a high fraction of methylated cytosines with a strong preference for CpG sites. The methylation pattern is highly similar among samples and selectively targets TEs rather than genes. A marked trend toward hypomethylation is observed for TEs located within a 1 kb distance from expressed genes, rather than segregated in TE-rich regions of the genome. Approximately 300 hypomethylated or unmethylated TEs are transcriptionally active, with higher expression levels in free-living mycelium compared to fruitbody. Indeed, multiple TE-enriched, copy number variant regions bearing a significant fraction of hypomethylated and expressed TEs are found almost exclusively in free-living mycelium. A reduction of DNA methylation, restricted to non-CpG sites and accompanied by an increase in TE expression, is observed upon treatment of free-living mycelia with 5-azacytidine.

Conclusions: Evidence derived from analysis of the T. melanosporum methylome indicates that a non-exhaustive, partly reversible, methylation process operates in truffles. This allows for the existence of hypomethylated, transcriptionally active TEs that are associated with copy number variant regions of the genome. Non-exhaustive TE methylation may reflect a role of active TEs in promoting genome plasticity and the ability to adapt to sudden environmental changes.
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http://dx.doi.org/10.1186/s13059-014-0411-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4165359PMC
July 2014

Alum and squalene-oil-in-water emulsion enhance the titer and avidity of anti-Aβ antibodies induced by multimeric protein antigen (1-11)E2, preserving the Igg1-skewed isotype distribution.

PLoS One 2014 1;9(7):e101474. Epub 2014 Jul 1.

Institute of Genetics and Biophysics, CNR, Napoli, Italy.

The development of active immunotherapy for Alzheimer's disease (AD) requires the identification of immunogens that can ensure a high titer antibody response toward Aβ, while minimizing the risks of adverse reactions. Multimeric protein (1-11)E2 induces a robust and persistent antibody response to Aβ in mice, when formulated in Freund's adjuvant. The goal of this translational study was to evaluate the immunogenicity of (1-11)E2 formulated in alum (Alhydrogel 2%), or in a squalene oil-in-water emulsion (AddaVax), or without adjuvant. A IgG1-skewed isotype distribution was observed for the anti-Aβ antibodies generated in mice immunized with either the non-adjuvanted or the adjuvanted vaccine, indicating that (1-11)E2 induces a Th2-like response in all tested conditions. Both Alhydrogel 2% and AddaVax enhanced the titer and avidity of the anti-Aβ response elicited by (1-11)E2. We conclude that (1-11)E2 is a promising candidate for anti-Aβ immunization protocols that include alum or squalene-oil-in-water emulsion, or no adjuvant.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0101474PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4077797PMC
October 2015

Integrative gene transfer in the truffle Tuber borchii by Agrobacterium tumefaciens-mediated transformation.

AMB Express 2014 29;4:43. Epub 2014 May 29.

Pasteur Cenci Bolognetti Foundation, c/o Department of Biology and Biotechnology "Charles Darwin", Sapienza University, Piazzale A. Moro 5, Rome, 00185, Italy ; Institute of Biology and Molecular Pathology, CNR, Rome, 00185, Italy.

Agrobacterium tumefaciens-mediated transformation is a powerful tool for reverse genetics and functional genomic analysis in a wide variety of plants and fungi. Tuber spp. are ecologically important and gastronomically prized fungi ("truffles") with a cryptic life cycle, a subterranean habitat and a symbiotic, but also facultative saprophytic lifestyle. The genome of a representative member of this group of fungi has recently been sequenced. However, because of their poor genetic tractability, including transformation, truffles have so far eluded in-depth functional genomic investigations. Here we report that A. tumefaciens can infect Tuber borchii mycelia, thereby conveying its transfer DNA with the production of stably integrated transformants. We constructed two new binary plasmids (pABr1 and pABr3) and tested them as improved transformation vectors using the green fluorescent protein as reporter gene and hygromycin phosphotransferase as selection marker. Transformants were stable for at least 12 months of in vitro culture propagation and, as revealed by TAIL- PCR analysis, integration sites appear to be heterogeneous, with a preference for repeat element-containing genome sites.
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http://dx.doi.org/10.1186/s13568-014-0043-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4052689PMC
June 2014

A high-performance thioredoxin-based scaffold for peptide immunogen construction: proof-of-concept testing with a human papillomavirus epitope.

Sci Rep 2014 Apr 22;4:4729. Epub 2014 Apr 22.

Department of Life Sciences, Biochemistry and Molecular Biology Unit, University of Parma, Italy.

Escherichia coli thioredoxin has been previously exploited as a scaffold for the presentation/stabilization of peptide aptamers as well as to confer immunogenicity to peptide epitopes. Here we focused on other key features of thioredoxin that are of general interest for the production of safer and more effective peptide immunogens, such as a high thermal stability, lack of cross-reactivity and a low-cost of production. We identified thioredoxin from the archaebacterium Pyrococcus furiosus (PfTrx) as a novel scaffold meeting all the above criteria. PfTrx is a highly thermostable and protease-resistant scaffold with a strong (poly)peptide solubilisation capacity. Anti-PfTrx antibodies did not cross-react with mouse, nor human thioredoxin. Untagged PfTrx bearing a previously identified HPV16-L2 peptide epitope was obtained in a >90% pure form with a one-step thermal purification procedure and effectively elicited the production of neutralizing anti-HPV antibodies. We thus propose PfTrx as a superior, general-purpose scaffold for the construction of safe, stable, and low-cost peptide immunogens.
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http://dx.doi.org/10.1038/srep04729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3994442PMC
April 2014