Real-time PCR for detection and quantification,and histological characterization of <Emphasis Type="Italic">Neonectria ditissima</Emphasis> in apple trees

Key message

We designed a pair of primers from a region of the β-tubulin gene to detect and quantify Neonectria ditissima in wood of some infected apple cultivars, and optimized light microscopy to study fungal-plant interactions.

Abstract

Neone ctria ditissima, the causal pathogen of fruit tree canker, is a sordariomycete fungus that affects apple orchards, especially in north-western Europe. To prevent serious disease epidemics, an accurate, rapid, and sensitive method for detection of N. ditissima is needed for pathogen identification. A quantitative real-time PCR (qPCR) assay was developed for both detection and quantification of this pathogen in infected apple cultivars. Several primer sets were designed from regions of the β-tubulin gene. One primer set passed several validation tests, and the melting curve confirmed species-specific amplification of the correct product. In addition, the N. ditissima biomass could be detected at variable amounts in samples from the infection sites of six different cultivars, with ‘Aroma’ having the lowest amount of N. ditissima biomass and ‘Elise’ the highest. To complement the qPCR results, tissue from detached shoots and 1-year-old trees of ‘Cox’s Orange Pippin’ (susceptible) and ‘Santana’ (partially resistant) was used in a histopathology study. In both detached shoots and trees, fungal hyphae were found in cells of all tissues. No qualitative differences in the anatomy of the infected samples were observed between the cultivars. In the detached shoot experiment, both cultivars were affected but differences in the rate of disease progression suggest that the partially resistant cultivar could resist the fungus longer. The qPCR assay developed in our study produced reproducible results and can be used for detection of N. ditissima in infected trees.

     

Meta-analysis Reveals Genome-Wide Significance at 15q13 for Nonsyndromic Clefting of Both the Lip and the Palate,and Functional Analyses Implicate GREM1 As a Plausible Causative Gene

Nonsyndromic orofacial clefts are common birth defects with multifactorial etiology. The most common type is cleft lip, which occurs with or without cleft palate (nsCLP and nsCLO, respectively). Although genetic components play an important role in nsCLP, the genetic factors that predispose to palate involvement are largely unknown. In this study, we carried out a meta-analysis on genetic and clinical data from three large cohorts and identified strong association between a region on chromosome 15q13 and nsCLP (P = 8.13×10⁻¹⁴ for rs1258763; relative risk (RR): 1.46, 95% confidence interval (CI): 1.32–1.61)) but not nsCLO (P = 0.27; RR: 1.09 (0.94–1.27)). The 5 kb region of strongest association maps downstream of Gremlin-1 (GREM1), which encodes a secreted antagonist of the BMP4 pathway. We show during mouse embryogenesis, Grem1 is expressed in the developing lip and soft palate but not in the hard palate. This is consistent with genotype-phenotype correlations between rs1258763 and a specific nsCLP subphenotype, since a more than two-fold increase in risk was observed in patients displaying clefts of both the lip and soft palate but who had an intact hard palate (RR: 3.76, CI: 1.47–9.61, P_diff<0.05). While we did not find lip or palate defects in Grem1-deficient mice, wild type embryonic palatal shelves developed divergent shapes when cultured in the presence of ectopic Grem1 protein (P = 0.0014). The present study identified a non-coding region at 15q13 as the second, genome-wide significant locus specific for nsCLP, after 13q31. Moreover, our data suggest that the closely located GREM1 gene contributes to a rare clinical nsCLP entity. This entity specifically involves abnormalities of the lip and soft palate, which develop at different time-points and in separate anatomical regions.     

Role of PKCtheta in macrophage-mediated immune response to <Emphasis Type="Italic">Salmonella typhimurium</Emphasis> infection in mice

Background

The serine/threonine protein kinase C (PKC) theta has been firmly implicated in T cell-mediated immunity. Because its role in macrophages has remained undefined, we employed PKCtheta-deficient (PKCtheta ^?/?) mice in order to investigate if PKCtheta plays a role in macrophage-mediated immune responses during bacterial infections.

Results

Our results demonstrate that PKCtheta plays an important role in host defense against the Gram-negative, intracellular bacterium Salmonella typhimurium, as reflected both by markedly decreased survival and a significantly enhanced number of bacteria in spleen and liver of PKCtheta ^?/? mice, when compared to wild-type mice. Of note, albeit macrophages do not express detectable PKCtheta, PKCtheta mRNA expression was found to be profoundly upregulated during the first hours of lipopolysaccharide (LPS)/interferon-gamma (IFNgamma)-, but not IL-4-mediated cell polarization conditions in vitro. Mechanistically, despite expressing normal levels of classically activated macrophage (CAM) markers, PKCtheta-deficient CAMs expressed significantly higher levels of the anti-inflammatory cytokine IL-10 in vivo and in vitro when challenged with S. typhimurium or LPS/IFNgamma. Neutralization of IL-10 recovered immune control to S. typhimurium infection in PKCtheta-deficient macrophages.

Conclusions

Taken together, our data provide genetic evidence that PKCtheta promotes a potent pro-inflammatory CAM phenotype that is instrumental to mounting protective anti-bacterial immunity. Mechanistically, PKCtheta exerts a host-protective role against S. typhimurium infection, and acts as an essential link between TLR4/IFNgammaR signaling and selective suppression of the anti-inflammatory cytokine IL-10 at the onset of CAM differentiation in the course of a bacterial infection.

     

Recognition of Intensive Valence and Arousal Affective States via Facial Electromyographic Activity in Young and Senior Adults

Background

Research suggests that interaction between humans and digital environments characterizes a form of companionship in addition to technical convenience. To this effect, humans have attempted to design computer systems able to demonstrably empathize with the human affective experience. Facial electromyography (EMG) is one such technique enabling machines to access to human affective states. Numerous studies have investigated the effects of valence emotions on facial EMG activity captured over the corrugator supercilii (frowning muscle) and zygomaticus major (smiling muscle). The arousal emotion, specifically, has not received much research attention, however. In the present study, we sought to identify intensive valence and arousal affective states via facial EMG activity.

Methods

Ten blocks of affective pictures were separated into five categories: neutral valence/low arousal (0VLA), positive valence/high arousal (PVHA), negative valence/high arousal (NVHA), positive valence/low arousal (PVLA), and negative valence/low arousal (NVLA), and the ability of each to elicit corresponding valence and arousal affective states was investigated at length. One hundred and thirteen participants were subjected to these stimuli and provided facial EMG. A set of 16 features based on the amplitude, frequency, predictability, and variability of signals was defined and classified using a support vector machine (SVM).

Results

We observed highly accurate classification rates based on the combined corrugator and zygomaticus EMG, ranging from 75.69% to 100.00% for the baseline and five affective states (0VLA, PVHA, PVLA, NVHA, and NVLA) in all individuals. There were significant differences in classification rate accuracy between senior and young adults, but there was no significant difference between female and male participants.

Conclusion

Our research provides robust evidences for recognition of intensive valence and arousal affective states in young and senior adults. These findings contribute to the successful future application of facial EMG for identifying user affective states in human machine interaction (HMI) or companion robotic systems (CRS).     

Use of Complementary and Alternative Medicine (CAM) as Part of the Oncological Treatment: Survey about Patients’ Attitude towards CAM in a University-Based Oncology Center in Germany

IntroductionTo understand if and which patients would be open-minded to Complementary and Alternative Medicine (CAM) use parallel to their oncological treatment. Moreover, we sought to determine which methods are most accepted and which are the primary motivators to use CAM.MethodsWe developed and anonymously conducted a questionnaire for patients in the oncology center (TU Munich). Questions focus on different CAM methods, previous experiences, and willingness to apply or use CAM when offered in a university-based setting.ResultsA total of 171 of 376 patients (37.4% women, 62.0% men, 0.6% unknown) participated. This corresponds to a return rate of 45%. Median age was 64 years (17–87 years). Of all participants, 15.2% used CAM during their oncological therapy; 32.7% have used it in the past. The majority (81.9%) was not using CAM during therapy; 55.5% have not used CAM in the past respectively. The analysis revealed a significant correlation between education and CAM use during therapy (r = 0.18; p = 0.02), and CAM use in the past (r = 0.17; p = 0.04). Of all patients using CAM during therapy, favored methods were food supplements (42.3%), vitamins/minerals (42.3%), massage (34.6%). Motivations are especially the reduction of side effect and stress, the positive effect of certain CAM-treatments on the immune system and tumor therapy. Results showed no difference between women and men. Most patients not having had any experience with CAM complain about the deficiency of information by their treating oncologist (31.4%) as well as missing treatment possibilities (54.3%).ConclusionSince many patients believe in study results demonstrating the efficacy of CAM, it stresses our task to develop innovative study protocols to investigate the outcomes of certain CAM on symptom reduction or other endpoints. Thus, prospective trials and innovative evidence-based treatment concepts to include CAM into high-end oncology is what patients demand and what a modern oncology center should offer.     

Systematic Investigation of Porphyrin‐Thiophene Conjugates for Ternary Bulk Heterojunction Solar Cells

A family of porphyrins and benzoporphyrins bearing phenyl, thiophenyl, or bithiophenyl groups at their meso‐positions are synthesized and systematically investigated for their potential use in bulk heterojunction solar cells (BHJ‐SCs). Comparative studies of these compounds show that the introduction of the thiophenyl and bithiophenyl groups, and the extension of the porphyrin π‐conjugated system significantly affect both photophysical and electrochemical properties. Binary conventional and ternary converted BHJ‐SCs based on these compounds are fabricated and studied. Results show that remarkable enhancement of the device efficiency is achieved by using the thiophene‐containing benzoporphyrin derivatives as additives for a poly(3‐hexylthiophene) (P3HT):phenyl‐C61‐butyric acid methyl ester blend in the inverted BHJ‐SCs. The optimum BHJ‐SC exhibits a maximum energy conversion efficiency of 4.3%, corresponding to 19% enhancement of the conversion efficiency as compared with the benchmark BHJ‐SCs.     

Manually and automatically produced pellet cultures of human primary chondrocytes: A comparative analysis

Cartilage defects are often associated with restriction of the locomotor system. New methods are required to investigate cartilage tissue and for the repair of cartilage tissue. 3D cultures are promising due to better simulation of in vivo conditions. The aim of this study was to provide a model system for studying cartilage tissue. We solved this problem by automated production of pellet cultures of human primary chondrocytes in media with and without antibiotics using the Biomek® Cell Workstation and consequent automated bioscreening with a high‐throughput screening system, and compared with the regular manual processes. The Biomek® Cell Workstation allows the cultivation of different cell types (suspensions cells and adherent cells) and 3D cell cultures (pellet cultures, alginate beads and spheroid cultures). The proliferation was analyzed by DNA quantification and compared with the EZ4U proliferation assay as a new tool for pellet cultures. The toxicity was evaluated by the detection of ubiquitous adenylate kinases. The proliferation increased from day 14 until day 35 and was associated with a decrease in the cytotoxicity. The comparative analysis showed similar results for manual and automated processes. We concluded that the manual methods can be replaced by automated processes (pellet manufacturing and screening), which would allow large‐scale procedures to support studies on cartilage regeneration.