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•HCMV-derived peptides for HLA-A∗03:01 and HLA-B∗15:01 haplotypes were identified via Ribo-seq, mass spectrometry, and machine learning.•Six novel immunogenic peptides were identified, establishing a framework for efficient detection of peptides from Ribo-seq data sets.

Dendritic cells (DCs) take up antigen in the periphery, migrate to secondary lymphoid organs, and present processed antigen fragments to adaptive immune cells and thus prime antigen-specific immunity. During local inflammation, recirculating monocytes are recruited from blood to the inflamed tissue, where they differentiate to macrophages and DCs. In this study, we found that monocytes showed high transporter associated with antigen processing (TAP)–dependent peptide compartmentalization and that after antigen pulsing, they were not able to efficiently stimulate antigen-specific T lymphocytes. Nevertheless, upon in vitro differentiation to monocyte-derived DCs, TAP-dependent peptide compartmentalization as well as surface major histocompatibility complex I turnover decreased and the cells efficiently restimulated T lymphocytes. Although TAP-dependent peptide compartmentalization decreased during DC differentiation, TAP expression levels increased. Furthermore, TAP relocated from early endosomes in monocytes to the endoplasmic reticulum (ER) and lysosomal compartments in DCs. Collectively, these data are compatible with the model that during monocyte-to-DC differentiation, the subcellular relocation of TAP and the regulation of its activity assure spatiotemporal separation of local antigen uptake and processing by monocytes and efficient T-lymphocyte stimulation by DCs.

Dendritic cells (DCs) take up antigen in the periphery, migrate to secondary lymphoid organs, and present processed antigen fragments to adaptive immune cells and thus prime antigen-specific immunity. During local inflammation, recirculating monocytes are recruited from blood to the inflamed tissue, where they differentiate to macrophages and DCs. In this study, we found that monocytes showed high transporter associated with antigen processing (TAP)–dependent peptide compartmentalization and that after antigen pulsing, they were not able to efficiently stimulate antigen-specific T lymphocytes. Nevertheless, upon in vitro differentiation to monocyte-derived DCs, TAP-dependent peptide compartmentalization as well as surface major histocompatibility complex I turnover decreased and the cells efficiently restimulated T lymphocytes. Although TAP-dependent peptide compartmentalization decreased during DC differentiation, TAP expression levels increased. Furthermore, TAP relocated from early endosomes in monocytes to the endoplasmic reticulum (ER) and lysosomal compartments in DCs. Collectively, these data are compatible with the model that during monocyte-to-DC differentiation, the subcellular relocation of TAP and the regulation of its activity assure spatiotemporal separation of local antigen uptake and processing by monocytes and efficient T-lymphocyte stimulation by DCs.

•Penton is an important immunodominant T-cell antigen in HAdV reactivation after HSCT.•Both penton- and hexon-specific T cells play an essential role in HAdV control.•Characteristics of antiviral T-cell response were similar albeit individually distinct.•Routine immunomonitoring should include penton-specific T cells.

Acyl homoserine lactones are synthesized by Pseudomonas aeruginosa as signaling molecules which control production of virulence factors and biofilm formation in a paracrine manner. We found that N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL), but not its 3-deoxo isomer or acyl-homoserine lactones with shorter fatty acids, induced the directed migration (chemotaxis) of human polymorphonuclear neutrophils (PMN) in vitro. By use of selective inhibitors a signaling pathway, comprising phosphotyrosine kinases, phospholipase C, protein kinase C, and mitogen-activated protein kinase C, could be delineated. In contrast to the well-studied chemokines complement C5a and interleukin 8, the chemotaxis did not depend on pertussis toxin-sensitive G proteins, indicating that 3OC12-HSL uses another signaling pathway. Strong evidence for the presence of a receptor for 3OC12-HSL on PMN was derived from uptake studies; by use of radiolabeled 3OC12-HSL, specific and saturable binding to PMN was seen. Taken together, our data provide evidence that PMN recognize and migrate toward a source of 3OC12-HSL (that is, to the site of a developing biofilm). We propose that this early attraction of PMN could contribute to prevention of biofilm formation.

ABSTRACTAcyl homoserine lactones are synthesized by Pseudomonas aeruginosaas signaling molecules which control production of virulence factors and biofilm formation in a paracrine manner. We found that N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL), but not its 3-deoxo isomer or acyl-homoserine lactones with shorter fatty acids, induced the directed migration (chemotaxis) of human polymorphonuclear neutrophils (PMN) in vitro. By use of selective inhibitors a signaling pathway, comprising phosphotyrosine kinases, phospholipase C, protein kinase C, and mitogen-activated protein kinase C, could be delineated. In contrast to the well-studied chemokines complement C5a and interleukin 8, the chemotaxis did not depend on pertussis toxin-sensitive G proteins, indicating that 3OC12-HSL uses another signaling pathway. Strong evidence for the presence of a receptor for 3OC12-HSL on PMN was derived from uptake studies; by use of radiolabeled 3OC12-HSL, specific and saturable binding to PMN was seen. Taken together, our data provide evidence that PMN recognize and migrate toward a source of 3OC12-HSL (that is, to the site of a developing biofilm). We propose that this early attraction of PMN could contribute to prevention of biofilm formation.

We generated and tested a set of cloning vectors designed to facilitate the production, purification and visualization of proteins in Dictyostelium discoideum. The vectors are derived from the Dictyostelium-E. colishuttle vector pDXA-3H (6.1 kb), which carries the origin of replication of the Ddhigh-copy-number plasmid, Ddp2, a high-copy-number E. coliplasmid origin of replication, an act6promoter driven G418 resistance cassette, the bacterial ampicillin resistance gene and an expression cassette. The new cloning vectors carry expression cassettes consisting of the strong constitutive actin-15 promoter, a translation start followed by a multiple cloning site, sequences for the addition of purification or visualization tags, and Dictyosteliumpolyadenylation and termination signals. Vectors designed to facilitate protein visualization in living Dictyosteliumcells contain either coding sequences for the cyan (CFP) or yellow (YFP) variants of green fluorescent protein (GFP). Versions of the vectors for the production of N- and C-terminal fusions with the fluorescent proteins were generated. To facilitate protein purification, vectors for the production of glutathione-S-transferase (GST) fusion proteins and Strep- or FLAG-affinity-tagged proteins were generated. Additionally, a vector for the production of His8-tagged proteins was generated, which has the G418-resistance cassette replaced by a hygromycin resistance cassette.

Plant diseases are a major threat to the world food supply, as up to 15% of production is lost to pathogens. In the past, disease control and the generation of resistant plant lines protected against viral, bacterial or fungal pathogens, was achieved using conventional breeding based on crossings, mutant screenings and backcrossing. Many approaches in this field have failed or the resistance obtained has been rapidly broken by the pathogens. Recent advances in molecular biotechnology have made it possible to obtain and to modify genes that are useful for generating disease resistant crops. Several strategies, including expression of pathogen-derived sequences or anti-pathogenic agents, have been developed to engineer improved pathogen resistance in transgenic plants. Antibody-based resistance is a novel strategy for generating transgenic plants resistant to pathogens. Decades ago it was shown that polyclonal and monoclonal antibodies can neutralize viruses, bacteria and selected fungi. This approach has been improved recently by the development of recombinant antibodies (rAbs). Crop resistance can be engineered by the expression of pathogen-specific antibodies, antibody fragments or antibody fusion proteins. The advantages of this approach are that rAbs can be engineered against almost any target molecule, and it has been demonstrated that expression of functional pathogen-specific rAbs in plants confers effective pathogen protection. The efficacy of antibody-based resistance was first shown for plant viruses and its application to other plant pathogens is becoming more established. However, successful use of antibodies to generate plant pathogen resistance relies on appropriate target selection, careful antibody design, efficient antibody expression, stability and targeting to appropriate cellular compartments.

We tested the hypothesis that membrane-anchored anti-viral antibodies can confer viral resistance to transgenic plants. A heterologous expression system was developed for plasma membrane targeting of anti-viral antibodies using mammalian transmembrane domains. A tobacco mosaic virus (TMV) neutralizing single-chain Fv antibody fragment (scFv24) was targeted to the endoplasmic reticulum and integrated into the plasma membrane of tobacco cells, using mammalian signal peptides and membrane receptor transmembrane domains. The human platelet-derived growth factor receptor (PDGFR) transmembrane domain or the T-cell receptor β-domain (TcRβ) transmembrane domain was fused to the C-terminus of TMV-specific scFv24 to target expression of scFv24 as an extracellularly facing plasma membrane protein. Western blot and ELISA analyses were carried out to confirm functional expression of the recombinant fusion proteins scFv24-PDGFR and scFv24-TcRβ in transgenic tobacco suspension cultures and transgenic plants. Immunofluorescence and electron microscopy showed that the TcRβ transmembrane domain targeted scFv24 to the tobacco plasma membrane. Bioassays of viral infection showed that transgenic tobacco plants expressing scFv24-TcRβ were resistant to TMV infection. These results demonstrated that membrane anchored anti-viral antibody fragments are functional, can be targeted to the plasma membrane in plantaand are a novel approach for engineering disease-resistant crops.

We compared the expression of a functional recombinant TMV‐specific full‐size antibody (rAb29) in both the apoplast and cytosol of tobacco plants and a single chain antibody fragment (scFv29), derived from rAb29, was expressed in the cytosol. Cloned heavy and light chain cDNAs of full‐size rAb29, which binds to TMV coat protein monomers, were integrated into the plant expression vector pSS. The full‐size rAb29 was expressed in the cytosol and targeted to the apoplast by including the original murine antibody leader sequences. Levels of functional full‐size rAb29 expression were high in the apoplast (up to 8.5 μg per gram leaf tissue), whereas cytosolic expression was low or at the ELISA detection limit. Sequences of the variable domains of rAb29 light and heavy chain were used to generate the single chain antibody scFv29, which was expressed in the periplasmic space of E.coli and showed the same binding specificity as full‐size rAb29. In addition, scFv29 was functionally expressed in the cytosol of tobacco plants and plant derived scFv29 maintained same binding specificity to TMV‐coat protein monomers as rAb29.

Studies of membrane electrical responses of isolated protoplasts to auxin have demonstrated the existence of elementary response chains to auxin at the plasma membrane, presently defined only by their uttermost ends. At one side, as demonstrated by several lines of evidence, the auxin perception unit involves proteins homologous to ZmER-abp1 (abp1), the most abundant auxin-binding protein from maize coleoptiles. At the other side, multiple ion transport proteins appear as targets of the auxin signal; the proton pump ATPase, an anion channel and potassium channels. We investigated early electrical responses to auxin at the plasma membrane of tobacco protoplasts. The work presented here will initially focus on abp1 and its functional role at the membrane. The C-terminus abp1 peptide (Pz151–163) was recently reported to modulate K+ currents at the plasma membrane of intact guard cells from broad bean [23] and induce plasma membrane hyperpolarisation of tobacco mesophyll protoplasts. These results further demonstrate that proteins involved in plasma membrane responses to auxin are related to maize abp1, and provide clues as to the region of the protein possibly involved in the interaction of abp1 with the plasma membrane. Secondly, this report concentrates on one of the targets of auxin, a voltage-dependent and ATP-regulated anion channel that we characterised on protoplasts from tobacco cell suspensions. This anion channel was specifically modulated by auxin, as already observed for the anion channel of guard cells [14]. Further work will be needed to assess if this auxin modulation involves a direct interaction between the hormone and the anion channel protein(s), or follows from the activation of a perception chain including abp1 homologues.

Inward rectifying potassium (K+in) channels play an important role in turgor regulation and ion uptake in higher plants. Here, we report a previously unrecognized feature of these proteins: K+inchannel C‐terminal polypeptides mediate channel protein interactions. Using a C‐terminal fragment of potato guard cell K+inchannel KST1 in a yeast two‐hybrid screen two novel putative K+inchannel proteins (SKT2 and SKT3) were identified by interaction of their C‐termini which contained a conserved domain (KHA). Interactions were confirmed by Western blot‐related assays utilizing K+inchannel C‐termini fused to green fluorescence protein. Although deletion of the KHA‐domain abolished these interactions, K+incurrents were still detectable by patch‐clamp measurements of insect cells expressing these KST1 mutants, indicating that formation of a functional channel does not depend on this C‐terminal domain.

Verapamil, nifedipine and bepridil, three antagonists of L‐type calcium channels in animal cells, were shown to induce direct inhibition of outward rectifying potassium current in Nicotiana tabacumcv. Xanthi protoplasts with IC50of 5 μM, 5 μM and 1 μM, respectively. In the outside‐out configuration, verapamil reduced the open probability of the ion channel responsible for the outward rectifying potassium conductance. Verapamil also blocked the outward rectifying potassium conductance in protoplasts from the N. tabacumcv. Bright Yellow cell line. Thus, studies using these molecules to demonstrate the involvement of calcium channels in plant physiological responses should be regarded with caution.

We evaluated the concept for protection of plants against virus infection based on the expression of single-chain Fv (scFv) fragments in the apoplasm or cytosol of transgenic plants. Cloned cDNA of a tobacco mosaic virus (TMV)-specific scFv antibody, which binds to intact virions, was integrated into the plant expression vector pSS and used for Agrobacterium-mediated transformation of Nicotiana tabacum cv. Xanthi-nc. Regenerated transgenic tobacco plants were analysed by northern blot, western blot and ELISA to assess expression and functionality of recombinant antibody (rAb) fragments. A significant increase of scFv levels in T1progeny was obtained for plants secreting apoplastic scFv antibodies but not for scFvs expressed in the cytosol. Bioassays revealed that T1progeny producing scFvs in different plant cell compartments showed different levels of resistance upon inoculation with TMV. The most dramatic reduction of necrotic local lesion numbers upon virus infection was observed in T1plants expressing scFv fragments in the cytosol. Infectivity could be reduced by more than 90%, despite the observation that protein expression levels for functional scFv antibodies were very low. Furthermore, upon inactivation of the N-resistance gene at elevated temperature, a significant portion of the T1progenies inhibited systemic virus spread, indicating that expression of TMV-specific cytosolic scFvs confers virus resistance in these transgenic plants. Moreover, inoculation of protoplasts isolated from transgenic and non-transgenic tobacco plants with TMV-RNA demonstrated that accumulation of virus particles is affected by cytosolic scFv expression.

AbstractDisease resistance in plant/pathogen interactions requires sensitive and specific recognition mechanisms for pathogen-derived signals in plants. Cultured parsley (Petroselinum crispum) cells respond to treatment with a crude cell wall preparation derived from the phytopathogenic fungus Phytophthora sojaewith transcriptional activation of the same set of defense-related genes as are activated in parsley leaves upon infection with fungal spores. A 13 amino acid core sequence (Pep-13) of a 42 kDa fungal cell wall glycoprotein was identified, which stimulates the same responses as the crude cell wall elicitor, namely macroscopic Ca2and H-influxes, effluxes of K- and Cl−ions, production of active oxygen species (oxidative burst), defense-related gene activation, and formation of antifungal phytoalexins. Using [125I]Tyr-Pep-13 as ligand in binding assays, a single-class high-affinity binding site in parsley microsomal membranes and protoplasts could be detected. Binding was specific, saturable, and reversible. By chemical crosslinking, a 91 kDa parsley plasma membrane protein was identified to be the receptor of the peptide elicitor. Isolation of this receptor protein involved in pathogen defense in plants is under way.

The error in lower Lehmann bounds to eigenvalues of self-adjoint problems is estimated from above by a constant multiple of the error in corresponding, upper Rayleigh-Ritz bounds. The constant involved is explicitly computable and monotonicallydecreasing in the dimension of the approximate eigenvalue problems. Asymptotically, the same inequality holds for the general Lehmann-Goerisch approach. Numerical examples are included in order to investigate the accordance of computed error quotients and theoretical bounds.