Candidate pathogenicity factor/effector proteins of 'Candidatus Phytoplasma solani' modulate plant carbohydrate metabolism, accelerate the ascorbate-glutathione cycle, and induce autophagosomes.

The pathogenicity of intracellular plant pathogenic bacteria is associated with the action of pathogenicity factors/effectors, but their physiological roles for most phytoplasma species, including 'Candidiatus Phytoplasma solani' are unknown. Six putative pathogenicity factors/effectors from six different strains of 'Ca. P. solani' were selected by bioinformatic analysis. The way in which they manipulate the host cellular machinery was elucidated by analyzing Nicotiana benthamiana leaves after Agrobacterium-mediated transient transformation with the pathogenicity factor/effector constructs using confocal microscopy, pull-down, and co-immunoprecipitation, and enzyme assays. Candidate pathogenicity factors/effectors were shown to modulate plant carbohydrate metabolism and the ascorbate-glutathione cycle and to induce autophagosomes. PoStoSP06, PoStoSP13, and PoStoSP28 were localized in the nucleus and cytosol. The most active effector in the processes studied was PoStoSP06. PoStoSP18 was associated with an increase in phosphoglucomutase activity, whereas PoStoSP28, previously annotated as an antigenic membrane protein StAMP, specifically interacted with phosphoglucomutase. PoStoSP04 induced only the ascorbate-glutathione cycle along with other pathogenicity factors/effectors. Candidate pathogenicity factors/effectors were involved in reprogramming host carbohydrate metabolism in favor of phytoplasma own growth and infection. They were specifically associated with three distinct metabolic pathways leading to fructose-6-phosphate as an input substrate for glycolysis. The possible significance of autophagosome induction by PoStoSP28 is discussed.

Understanding pandemic resilience: a mixed-methods exploration of burdens, resources, and determinants of good or poor well-being among Austrian psychotherapists.

Introduction: The COVID-19 pandemic has exacerbated the mental health burden on the general population, resulting in increased demands on mental healthcare professionals, including psychotherapists. This cross-sectional study assessed the challenges and resources encountered by 513 psychotherapists based on an online survey conducted between April and May 2022. Methods: Qualitative methods content analysis of written reports was employed to investigate the emerging challenges and sources of support during the pandemic. A comparative analysis of burdens, resources, sociodemographic factors and daily physical activity was conducted to discern patterns of good and poor well-being. Results: The predominant burden identified was mental health-related issues, followed by global crises and government-imposed restrictions to mitigate virus transmission. Essential resources encompassed social connections, mindfulness, work satisfaction, and internal processes. Notably, psychotherapists demonstrating good well-being were older, more physically active, had a lower proportion of females, were employed in private practices rather than in institutionalized settings, had more years of professional experience and treated more patients weekly than their counterparts with poor well-being. Furthermore, they exhibited greater optimism, health focus, and satisfaction with their coping methods. Discussion: These findings can help develop support systems, policies, and educational programs to better support mental health professionals during global crises and offer strategies for individual practitioners to maintain their well-being.

Effects of insecticides and repellents on the spread of 'Candidatus Phytoplasma solani' under laboratory and field conditions.

Recent outbreaks of 'Candidatus Phytoplasma solani' resulted in severe losses in potatoes, vegetable crops and grapevines in certain regions of Austria and constituted a major challenge for seed potato production. Therefore, the effects of various insecticides and insect deterrents on pathogen spread were studied both in laboratory and field experiments from 2018 to 2021. In laboratory transmission experiments, field captured Hyalesthes obsoletus were caged on differently treated Catharanthus roseus for five days. The insecticides lambda-cyhalothrin, deltamethrin, esfenvalerate, acetamiprid and chlorpyriphos showed the most rapid impact on insect survival and fully prevented phytoplasma transmission. The particle film forming products kaolin and diatomaceous earth had some effect. A transfer of the promising laboratory results to potato fields, however, was achieved to a limited extent only. Treatments with pyrethroids and acetamiprid every 8-10 days over the flight period of H. obsoletus roughly halved the number of symptomatic plants and tubers in case of moderately susceptible varieties and moderate infection pressure. In the event of susceptible varieties and high disease pressure, treatment effects were hardy discernible. In practical terms, the experiments indicate that insecticide applications alone are not sufficient to mitigate the disease. Spraying of diatomaceous earth and mineral oil did not affect disease incidence in the field. Supplementary Information: The online version contains supplementary material available at 10.1007/s41348-023-00768-y.

Geographical and Temporal Diversity of 'Candidatus Phytoplasma solani' in Wine-Growing Regions in Slovenia and Austria.

As the causal agent of the grapevine yellows disease Bois noir, 'Candidatus Phytoplasma solani' has a major economic impact on grapevines. To improve the control of Bois noir, it is critical to understand the very complex epidemiological cycles that involve the multiple "Ca. P. solani" host plants and insect vectors, of which Hyalesthes obsoletus is the most important. In the present study, multiple genotyping of the tuf, secY, stamp, and vmp1 genes was performed. This involved archived grapevine samples that were collected during an official survey of grapevine yellows throughout the wine-growing regions of Slovenia (from 2003 to 2016), plus samples from Austrian grapevines, stinging nettle, field bindweed, and insect samples (collected from 2012 to 2019). The data show that the tuf-b2 type of the tuf gene has been present in eastern Slovenia since at least 2003. The hypotheses that the occurrence of the haplotypes varies due to the geographical position of Slovenia on the Italian-Slovenian Karst divide and that the haplotypes are similar between Slovenian and Austrian Styria were confirmed. The data also show haplotype changes for host plants and H. obsoletus associated with 'Ca. P. solani,' which might be linked to new epidemiological cycles of this phytoplasma that involve not just new plant sources and new insect vectors, but also climate and land-use changes.

Vector transmission and epidemiology of 'Candidatus Phytoplasma pyri' in Austria and identification of Cacopsylla pyrisuga as new pathogen vector.

Pear decline, induced by the phytoplasma 'Candidatus Phytoplasma pyri', transmitted by pear psyllids, is one of the most devastating diseases on Pyrus communis in Europe and North America. Investigations of pear psyllids in 4 pear orchards in lower Austria showed the presence of Cacopsylla pyri, C. pyricola and C. pyrisuga at all locations. PCR analyses revealed overall phytoplasma infection rates for C. pyri of 5.4%, for C. pyricola, of 4.6%, for C. pyrisuga remigrants of 9.6% and for C. pyrisuga emigrants of 0%. The rates of PCR-positive C. pyri and C. pyricola individuals varied greatly in the course of the year, and the highest infection rates were observed in late summer, autumn and in late winter. In transmission experiments with healthy pear seedlings, winterform individuals of C. pyri and C. pyricola transmitted the pathogen to 19.2% (5 out of 26) and 4.8% (2 out of 41) of the test plants, respectively. The vectoring ability of C. pyrisuga was experimentally proven for the first time, and in transmission experiments with remigrants, 9.5% (2 out of 21) of the pear seedlings were infected. Our data indicate a significant risk of pathogen transmission in pear orchards during the greater part of the year, especially in late winter, early spring and autumn. Multilocus sequence analysis by aid of the genes aceF and imp allowed the discrimination between 15 phytoplasma types. Three so far undescribed aceF genotypes and four undescribed imp genotypes were identified. Supplementary Information: The online version contains supplementary material available at 10.1007/s41348-021-00526-y.

Optimization of the Alizarin Red S Assay by Enhancing Mineralization of Osteoblasts.

The alizarin red S assay is considered the gold standard for quantification of osteoblast mineralization and is thus widely used among scientists. However, there are several restrictions to this method, e.g., moderate sensitivity makes it difficult to uncover slight but significant effects of potentially clinically relevant substances. Therefore, an adaptation of the staining method is appropriate and might be obtained by increasing the mineralization ability of osteoblasts. In this study, cell culture experiments with human (SaOs-2) and murine (MC3T3-E1) osteoblasts were performed under the addition of increasing concentrations of calcium chloride (1, 2.5, 5, and 10 mM) or calcitonin (1, 2.5, 5, and 10 nM). After three or four weeks, the mineralization matrix was stained with alizarin red S and the concentration was quantified photometrically. Only calcium chloride was able to significantly increase mineralization, and therefore enhanced the sensitivity of the alizarin red S staining in a dose-dependent manner in both osteoblastic cell lines as well as independent of the cell culture well surface area. This cost- and time-efficient optimization enables a more sensitive analysis of potentially clinically relevant substances in future bone research.

Differential Response of Grapevine to Infection with 'Candidatus Phytoplasma solani' in Early and Late Growing Season through Complex Regulation of mRNA and Small RNA Transcriptomes.

Bois noir is the most widespread phytoplasma grapevine disease in Europe. It is associated with 'Candidatus Phytoplasma solani', but molecular interactions between the causal pathogen and its host plant are not well understood. In this work, we combined the analysis of high-throughput RNA-Seq and sRNA-Seq data with interaction network analysis for finding new cross-talks among pathways involved in infection of grapevine cv. Zweigelt with 'Ca. P. solani' in early and late growing seasons. While the early growing season was very dynamic at the transcriptional level in asymptomatic grapevines, the regulation at the level of small RNAs was more pronounced later in the season when symptoms developed in infected grapevines. Most differentially expressed small RNAs were associated with biotic stress. Our study also exposes the less-studied role of hormones in disease development and shows that hormonal balance was already perturbed before symptoms development in infected grapevines. Analysis at the level of communities of genes and mRNA-microRNA interaction networks revealed several new genes (e.g., expansins and cryptdin) that have not been associated with phytoplasma pathogenicity previously. These novel actors may present a new reference framework for research and diagnostics of phytoplasma diseases of grapevine.

Cystatin F is a biomarker of prion pathogenesis in mice.

Misfolding of the cellular prion protein (PrPC) into the scrapie prion protein (PrPSc) results in progressive, fatal, transmissible neurodegenerative conditions termed prion diseases. Experimental and epidemiological evidence point toward a protracted, clinically silent phase in prion diseases, yet there is no diagnostic test capable of identifying asymptomatic individuals incubating prions. In an effort to identify early biomarkers of prion diseases, we have compared global transcriptional profiles in brains from pre-symptomatic prion-infected mice and controls. We identified Cst7, which encodes cystatin F, as the most strongly upregulated transcript in this model. Early and robust upregulation of Cst7 mRNA levels and of its cognate protein was validated in additional mouse models of prion disease. Surprisingly, we found no significant increase in cystatin F levels in both cerebrospinal fluid or brain parenchyma of patients with Creutzfeldt-Jakob disease compared to Alzheimer's disease or non-demented controls. Our results validate cystatin F as a useful biomarker of early pathogenesis in experimental models of prion disease, and point to unexpected species-specific differences in the transcriptional responses to prion infections.

Mining the human autoantibody repertoire: isolation of potent IL17A-neutralizing monoclonal antibodies from a patient with thymoma.

Anti-cytokine autoantibodies have been widely reported to be present in human plasma, both in healthy subjects and in patients with underlying autoimmune conditions, such as autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) or thymic epithelial neoplasms. While often asymptomatic, they can cause or facilitate a wide range of diseases including opportunistic infections. The potential therapeutic value of specific neutralizing anti-cytokine autoantibodies has not been thoroughly investigated. Here we used mammalian cell display to isolate IL17A-specific antibodies from a thymoma patient with proven high-titer autoantibodies against the same. We identified 3 distinct clonotypes that efficiently neutralized IL17A in a cell-based in vitro assay. Their potencies were comparable to those of known neutralizing antibodies, including 2, AIN457 (secukinumab) and ixekizumab that are currently in clinical development for the treatment of various inflammatory disorders. These data clearly demonstrate that the human autoantibody repertoire can be mined for antibodies with high therapeutic potential for clinical development.

Metal fluoride-based transparent nanocomposites with low refractive indices.

The utilisation of magnesium and aluminium fluoride nanoparticles in the preparation of transparent composites leading to materials with superior properties was investigated. Nanoscopic magnesium and aluminium fluoride has been prepared by the fluorolytic sol-gel route from the alkoxides and was surface modified by the reaction with trifluoroacetic acid or perfluorobutyric acid. IR spectroscopic experiments of the xerogels and crystal structure analysis of a trinuclear [Mg3(µ3F)(µ-TFA)6(OCH3)2(py)](3-) cluster unit indicate that the carboxylate group is bound to the particle surface in a monodentate or bidentate bridging fashion. These particles were successfully incorporated into acrylate polymers with up to 40 wt% content to give fully transparent material. Ellipsometry and m-line measurements of thin films show the reduction of the refractive index of composite films with increasing metal fluoride filler content.

Low-affinity B cells transport viral particles from the lung to the spleen to initiate antibody responses.

The lung is an important entry site for pathogens; its exposure to antigens results in systemic as well as local IgA and IgG antibodies. Here we show that intranasal administration of virus-like particles (VLPs) results in splenic B-cell responses with strong local germinal-center formation. Surprisingly, VLPs were not transported from the lung to the spleen in a free form but by B cells. The interaction between VLPs and B cells was initiated in the lung and occurred independently of complement receptor 2 and Fcγ receptors, but was dependent upon B-cell receptors. Thus, B cells passing through the lungs bind VLPs via their B-cell receptors and deliver them to local B cells within the splenic B-cell follicle. This process is fundamentally different from delivery of blood or lymph borne particulate antigens, which are transported into B cell follicles by binding to complement receptors on B cells.

Universal vaccine against influenza virus: linking TLR signaling to anti-viral protection.

A vaccine protecting against all influenza strains is a long-sought goal, particularly for emerging pandemics. As previously shown, vaccines based on the highly conserved extracellular domain of M2 (M2e) may protect against all influenza A strains. Here, we demonstrate that M2e-specific monoclonal antibodies (mAbs) protect mice from a lethal influenza infection. To be protective, antibodies had to be able to bind to Fc receptors and fix complement. Furthermore, mAbs of IgG2c isotype were protective in mice, while antibodies of identical specificity, but of the IgG1 isotype, failed to prevent disease. These findings readily translated into vaccine design. A vaccine targeting M2 in the absence of a toll-like receptor (TLR) 7 ligand primarily induced IgG1, whilst the same vaccine linked to a TLR7 ligand yielded high levels of IgG2c antibodies. Although both vaccines protected mice from a lethal challenge, mice treated with the vaccine containing a TLR7 ligand showed significantly lower morbidity. In accordance with these findings, vaccination of TLR7(-/-) mice with a vaccine containing a TLR7 ligand did not result in protection from a lethal challenge. Hence, the innate immune system is required to direct isotype switching toward the more protective IgG2a/c antibodies.

Cuing consumerism: situational materialism undermines personal and social well-being.

Correlational evidence indicates that materialistic individuals experience relatively low levels of well-being. Across four experiments, we found that situational cuing can also trigger materialistic mind-sets, with similarly negative personal and social consequences. Merely viewing desirable consumer goods resulted in increases in materialistic concerns and led to heightened negative affect and reduced social involvement (Experiment 1). Framing a computer task as a "Consumer Reaction Study" led to a stronger automatic bias toward values reflecting self-enhancement, compared with framing the same task as a "Citizen Reaction Study" (Experiment 2). Consumer cues also increased competitiveness (Experiment 3) and selfishness in a water-conservation dilemma (Experiment 4). Thus, the costs of materialism are not localized only in particularly materialistic people, but can also be found in individuals who happen to be exposed to environmental cues that activate consumerism-cues that are commonplace in contemporary society.

Different binding modes of free and carrier-protein-coupled nicotine in a human monoclonal antibody.

Nicotine is the principal addictive component of tobacco. Blocking its passage from the lung to the brain with nicotine-specific antibodies is a promising approach for the treatment of smoking addiction. We have determined the crystal structure of nicotine bound to the Fab fragment of a fully human monoclonal antibody (mAb) at 1.85 Å resolution. Nicotine is almost completely (>99%) buried in the interface between the variable domains of heavy and light chains. The high affinity of the mAb is the result of a charge-charge interaction, a hydrogen bond, and several hydrophobic contacts. Additionally, similarly to nicotinic acetylcholine receptors in the brain, two cation-π interactions are present between the pyrrolidine charge and nearby aromatic side chains. The selectivity of the mAb for nicotine versus cotinine, which is the major metabolite of nicotine and differs in only one oxygen atom, is caused by steric constraints in the binding site. The mAb was isolated from B cells of an individual immunized with a nicotine-carrier protein conjugate vaccine. Surprisingly, the nicotine was bound to the Fab fragment in an orientation that was not compatible with binding to the nicotine-carrier protein conjugate. The structure of the Fab fragment in complex with the nicotine-linker derivative that was used for the production of the conjugate vaccine revealed a similar position of the pyridine ring of the nicotine moiety, but the pyrrolidine ring was rotated by about 180°. This allowed the linker part to reach to the Fab surface while high-affinity interactions with the nicotine moiety were maintained.

Inorganic-organic nanocomposites based on sol-gel derived magnesium fluoride.

Monodispersed magnesium fluoride nanoparticles are utilized for the first time to prepare transparent inorganic-organic nanocomposite materials with improved mechanical properties. The fluorolytic sol-gel synthesis route has been modified for the preparation of monodispersed magnesium fluoride nanoparticles with a size of 2-3 nm. MgF(2) particles are effectively stabilised against agglomeration by phosphonic acids, which strongly bind to the particles and lead to an increased compatibility of the inorganic particles with the organic polymers. This way, highly transparent nanocomposite materials with up to 20 wt% magnesium fluoride in different acrylates are obtained, featuring high dispersion of MgF(2) particles in the polymer matrix and an increased hardness by the factor of 2. The nature of interaction between phosphonic acids and magnesium fluoride is thoroughly investigated by IR and NMR showing a monodentate coordination of phosphonates to the particle's surface.

Mechanisms of allergen-specific desensitization.

BACKGROUND: Allergen-specific desensitization (SIT) is the most effective therapy for allergies. Although allergen-specific antibodies have an important role in the process, mechanisms of IgG-mediated inhibition of allergic reactions are not well defined. OBJECTIVE: We investigated mechanisms by which SIT-induced allergen-specific IgGs inhibit allergic reactions. METHODS: We generated mAbs that recognize 3 nonoverlapping epitopes of the major cat allergen Fel d 1. Each of the mAbs was produced as an IgE and different IgG isotype. RESULTS: IgEs against 2 nonoverlapping epitopes on Fel d 1 are necessary and sufficient to sensitize mast cells for maximal FcepsilonRI signaling and degranulation on exposure to monomeric Fel d 1. IgE antibodies of a third specificity did not further increase mast cell degranulation, indicating that formation of large FcepsilonRI clusters are not required to induce maximal activation of mast cells. A single IgG that was specific for an epitope different from those recognized by the IgEs was a potent inhibitor of Fel d 1-mediated mast cell activation in vitro and in vivo. This inhibition required Fcgamma receptor-IIB. In human beings, IgGs of a single specificity were able to block degranulation of basophils from individuals with cat allergy. The inhibitory potential of these antibodies increased when larger allergen-IgG complexes were formed. CONCLUSIONS: These data reconcile conflicting theories in the literature and might explain the reason IgE levels do not necessarily decrease during therapy, despite clinical efficacy. These findings have important implications for vaccine design.

Innate signaling regulates cross-priming at the level of DC licensing and not antigen presentation.

Innate stimuli, such as TLR ligands, are known to greatly facilitate cross-priming. Currently it is unclear whether innate stimuli enhance cross-priming at the level of cross-presentation or at the level of T-cell priming. In this study, we addressed this question by measuring cross-presentation as well as cross-priming by virus-like particles (VLP) displaying peptide p33 derived of lymphocytic choriomeningitis virus. Innate stimuli were varied by either packaging different TLR ligands into virus-like particles or using mice deficient in two key molecules of TLR-signaling, namely the adaptor molecule MyD88 as well as IFN-alpha/beta receptor. While efficient cross-presentation occurred despite strongly reduced activation of DC in the absence of TLR ligand-mediated signals, T-cell priming was abolished. Thus, innate stimuli regulate cross-priming at the level of DC licensing for T-cell activation and not antigen presentation.

Cutting edge: limited specialization of dendritic cell subsets for MHC class II-associated presentation of viral particles.

Dendritic cells (DCs) are the most important APC. It was recently reported that there is a dichotomy for Ag presentation by DC subsets; exogenous Ags reach the MHC class I pathway, but not the MHC class II pathway, in CD8(+) DCs, whereas CD8(-) DCs only process Ags for the MHC class II pathway. In this study, we used virus-like particles (VLPs) to show that CD8(+) and CD8(-) DCs efficiently capture and process VLPs for presentation in association with MHC class II in vivo. In contrast, CD8(+) DCs, but not CD8(-) DCs, cross presented VLP-derived peptides. This pattern was changed in an FcgammaR-dependent fashion in the presence of VLP-specific Abs, because under those conditions both DC subsets failed to efficiently cross present. Thus, the presentation of viral particles to CD4(+) T cells is not restricted to distinct DC subsets, whereas the presentation of viral particles to CD8(+) T cells is limited to CD8(+) DCs.

Prophylactic and therapeutic activity of fully human monoclonal antibodies directed against influenza A M2 protein.

Influenza virus infection is a prevalent disease in humans. Antibodies against hemagglutinin have been shown to prevent infection and hence hemagglutinin is the major constituent of current vaccines. Antibodies directed against the highly conserved extracellular domain of M2 have also been shown to mediate protection against Influenza A infection in various animal models. Active vaccination is generally considered the best approach to combat viral diseases. However, passive immunization is an attractive alternative, particularly in acutely exposed or immune compromized individuals, young children and the elderly. We recently described a novel method for the rapid isolation of natural human antibodies by mammalian cell display. Here we used this approach to isolate human monoclonal antibodies directed against the highly conserved extracellular domain of the Influenza A M2 protein. The identified antibodies bound M2 peptide with high affinities, recognized native cell-surface expressed M2 and protected mice from a lethal influenza virus challenge. Moreover, therapeutic treatment up to 2 days after infection was effective, suggesting that M2-specific monoclonals have a great potential as immunotherapeutic agents against Influenza infection.

Displaying Fel d1 on virus-like particles prevents reactogenicity despite greatly enhanced immunogenicity: a novel therapy for cat allergy.

Allergen-specific desensitization is the only disease-modifying therapy currently available for the treatment of allergies. These therapies require application of allergen over several years and some may induce life-threatening anaphylactic reactions. An ideal vaccine for desensitization should be highly immunogenic and should alleviate allergic symptoms upon few injections while being nonreactogenic. We describe such a vaccine for the treatment of cat allergy, consisting of the major cat allergen Fel d1 coupled to bacteriophage Qbeta-derived virus-like particles (Qbeta-Fel d1). Qbeta-Fel d1 was highly immunogenic, and a single vaccination was sufficient to induce protection against type I allergic reactions. Allergen-specific immunoglobulin G antibodies were shown to be the critical effector molecules and alleviated symptoms by two distinct mechanisms. Although allergen-induced systemic basophil degranulation was inhibited in an FcgammaRIIb-dependent manner, inhibition of local mast cell degranulation in tissues occurred independently of FcgammaRIIb. In addition, treatment with Qbeta-Fel d1 abolished IgE memory responses upon antigen recall. Despite high immunogenicity, the vaccine was essentially nonreactogenic and vaccination induced neither local nor systemic anaphylactic reactions in sensitized mice. Moreover, Qbeta-Fel d1 did not induce degranulation of basophils derived from human volunteers with cat allergies. These data suggest that vaccination with Qbeta-Fel d1 may be a safe and effective treatment for cat allergy.