Identification of quantitative trait loci contributing to yield and seed quality parameters under terminal drought in barley advanced backcross lines

Drought is a major limiting factor for barley production, especially in the primary areas of its cultivation. Wild barley represents a major source of favourable alleles for increasing the genetic variation for multiple traits including resistance to both biotic and abiotic stresses. We used advanced backcross quantitative trait locus (AB-QTL) analysis of a BC3-doubled haploid population developed between the cultivated parent Brenda (Hordeum vulgare ssp. vulgare) and the wild accession HS584 (H. vulgare ssp. spontaneum) to study the contribution of wild barley in improving various agronomic and seed quality traits under post-anthesis drought. The experiment was carried out at two different locations (IPK, Gatersleben and Nordsaat, Böhnshausen) and terminal drought was imposed by withholding water or spraying with potassium iodide at 10 days after flowering under greenhouse or field conditions, respectively. QTL analysis indicated that wild barley contributed favourably to most of the traits studied under both control and drought conditions. A total of seven hot-spot QTL regions with co-localizing QTL for various traits harboured more than 80 % of the stable QTL detected in the present study. For yield and thousand-grain weight and their respective drought tolerance indices, most of the QTL were derived from Brenda. On the other hand, for traits like seed length and seed nitrogen content, all the QTL were contributed by HS584, the parent having higher trait value. A significantly reduced carbon/nitrogen (C/N) ratio in the selected contrasting inferior lines compared to superior ones suggests that C/N ratio could be a potential parameter for screening not just seed quality parameters but also grain weight performance under terminal drought.     

Involvement of two type 2C protein phosphatases BcPtc1 and BcPtc3 in the regulation of multiple stress tolerance and virulence of Botrytis cinerea

Type 2C Ser/Thr phosphatases (PP2Cs) are involved in various cellular processes in many eukaryotes, but little has been known about their functions in filamentous fungi. Botrytis cinerea contains four putative PP2C genes, named BcPTC1, ‐3, ‐5, and ‐6. Biological functions of these genes were analysed by gene deletion and complementation. While no phenotypes aberrant from the wild type were observed with mutants of BcPTC5 and BcPTC6, mutants of BcPTC1 and BcPTC3 had reduced hyphal growth, increased conidiation, and impaired sclerotium development. Additionally, BcPTC1 and BcPTC3 mutants exhibited increased sensitivity to osmotic and oxidative stresses, and to cell wall degrading enzymes. Both mutants exhibited dramatically decreased virulence on host plant tissues. All of the defects were restored by genetic complementation of the mutants with wild‐type BcPTC1 and BcPTC3 respectively. Different from what is known in Saccharomyces cerevisiae, BcPtc3, but not BcPtc1, negatively regulates phosphorylation of BcSak1 (the homologue of S. cerevisiae Hog1) in B. cinerea, although both BcPTC1 and BcPTC3 were able to rescue the growth defects of a yeast PTC1 deletion mutant under various stress conditions. These results demonstrated that BcPtc1 and BcPtc3 play important roles in the regulation of multiple stress tolerance and virulence of B. cinerea.     

An Abscisic Acid-Independent Oxylipin Pathway Controls Stomatal Closure and Immune Defense in Arabidopsis

Plant stomata function in innate immunity against bacterial invasion and abscisic acid (ABA) has been suggested to regulate this process. Using genetic, biochemical, and pharmacological approaches, we demonstrate that (i) the Arabidopsis thaliana nine-specific-lipoxygenase encoding gene, LOX1, which is expressed in guard cells, is required to trigger stomatal closure in response to both bacteria and the pathogen-associated molecular pattern flagellin peptide flg22; (ii) LOX1 participates in stomatal defense; (iii) polyunsaturated fatty acids, the LOX substrates, trigger stomatal closure; (iv) the LOX products, fatty acid hydroperoxides, or reactive electrophile oxylipins induce stomatal closure; and (v) the flg22-mediated stomatal closure is conveyed by both LOX1 and the mitogen-activated protein kinases MPK3 and MPK6 and involves salicylic acid whereas the ABA-induced process depends on the protein kinases OST1, MPK9, or MPK12. Finally, we show that the oxylipin and the ABA pathways converge at the level of the anion channel SLAC1 to regulate stomatal closure. Collectively, our results demonstrate that early biotic signaling in guard cells is an ABA-independent process revealing a novel function of LOX1-dependent stomatal pathway in plant immunity.     

Raccoons (Procyon lotor) in Germany as potential reservoir species for Lyssaviruses

Raccoons can be found almost everywhere in Germany since their first successful introduction in 1934. Although the animal is a well-known reservoir species for rabies in the USA, during the last European fox rabies epizootic, only a few rabid raccoons were reported from Germany. In recent years, the raccoon population density has increased tremendously, especially in (semi) urban settings. Presently, Germany is free of terrestrial wildlife rabies. To assess the potential risk that the raccoon population in Germany could act as a reservoir species upon reemergence of rabies, the susceptibility of the local raccoon population was investigated. Wild-caught animals were inoculated with the most likely lyssavirus variants to infect the local population. It was shown that the raccoons were fully susceptible for a dog and raccoon rabies virus isolate. Also, five of six raccoons inoculated with a fox rabies virus isolate showed clinical signs. However, none of the raccoons infected with European Bat Lyssavirus type 1 succumbed to rabies; meanwhile, all these raccoons seroconverted. It is concluded that the highest risk for the raccoon population in Germany to become infected with lyssaviruses is through the importation of rabies infected dogs.     

Lactobacillus paracasei DSMZ16671 Reduces Mutans Streptococci: A Short-Term Pilot Study

Reducing the burden of pathogenic mutans streptococci is a goal of oral health. Lactobacillus paracasei DSMZ16671, even after heat-killing, specifically co-aggregates mutans streptococci in vitro and retains this activity in human saliva. In rats, it reduces mutans streptococcal colonization of teeth and caries scores. This pilot study sought to assess the potential of heat-killed L. paracasei DSMZ16671 (pro-t-action^®) to reduce levels of salivary mutans streptococci in humans, using sugar-free candies as a delivery vehicle. A randomized, placebo-controlled, double-blind in vivo study of three groups examined the short-term effect of sugar-free candies containing 0 (placebo), 1, or 2 mg/candy piece of heat-killed L. paracasei DSMZ16671 on the levels of salivary mutans streptococci determined before and after consumption of the candies. The candies were consumed 4 times during 1.5 consecutive days. Compared to the placebo group, the test groups’ saliva had significantly reduced mutans streptococci as an immediate effect. These results suggest the use of heat-killed L. paracasei DSMZ16671 in suckable candies as a method to reduce mutans streptococci in the mouth and, thereby, caries risk. We think this a new concept and strategy for caries prevention and management.     

FAS-Dependent Cell Death in α-Synuclein Transgenic Oligodendrocyte Models of Multiple System Atrophy

Multiple system atrophy is a parkinsonian neurodegenerative disorder. It is cytopathologically characterized by accumulation of the protein p25α in cell bodies of oligodendrocytes followed by accumulation of aggregated α-synuclein in so-called glial cytoplasmic inclusions. p25α is a stimulator of α-synuclein aggregation, and coexpression of α-synuclein and p25α in the oligodendroglial OLN-t40-AS cell line causes α-synuclein aggregate-dependent toxicity. In this study, we investigated whether the FAS system is involved in α-synuclein aggregate dependent degeneration in oligodendrocytes and may play a role in multiple system atrophy. Using rat oligodendroglial OLN-t40-AS cells we demonstrate that the cytotoxicity caused by coexpressing α-synuclein and p25α relies on stimulation of the death domain receptor FAS and caspase-8 activation. Using primary oligodendrocytes derived from PLP-α-synuclein transgenic mice we demonstrate that they exist in a sensitized state expressing pro-apoptotic FAS receptor, which makes them sensitive to FAS ligand-mediated apoptosis. Immunoblot analysis shows an increase in FAS in brain extracts from multiple system atrophy cases. Immunohistochemical analysis demonstrated enhanced FAS expression in multiple system atrophy brains notably in oligodendrocytes harboring the earliest stages of glial cytoplasmic inclusion formation. Oligodendroglial FAS expression is an early hallmark of oligodendroglial pathology in multiple system atrophy that mechanistically may be coupled to α-synuclein dependent degeneration and thus represent a potential target for protective intervention.     

CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory,GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.     

Are Treponema pallidum Specific Rapid and Point-of-Care Tests for Syphilis Accurate Enough for Screening in Resource Limited Settings? Evidence from a Meta-Analysis

Background

Rapid and point-of-care (POC) tests for syphilis are an invaluable screening tool, yet inadequate evaluation of their diagnostic accuracy against best reference standards limits their widespread global uptake. To fill this gap, a systematic review and meta-analysis was conducted to evaluate the sensitivity and specificity of rapid and POC tests in blood and serum samples against Treponema pallidum (TP) specific reference standards.

Methods

Five electronic databases (1980–2012) were searched, data was extracted from 33 articles, and Bayesian hierarchical models were fit.

Results

In serum samples, against a TP specific reference standard point estimates with 95% credible intervals (CrI) for the sensitivities of popular tests were: i) Determine, 90.04% (80.45, 95.21), ii) SD Bioline, 87.06% (75.67, 94.50), iii) VisiTect, 85.13% (72.83, 92.57), and iv) Syphicheck, 74.48% (56.85, 88.44), while specificities were: i) Syphicheck, 99.14% (96.37, 100), ii) Visitect, 96.45% (91.92, 99.29), iii) SD Bioline, 95.85% (89.89, 99.53), and iv) Determine, 94.15% (89.26, 97.66). In whole blood samples, sensitivities were: i) Determine, 86.32% (77.26, 91.70), ii) SD Bioline, 84.50% (78.81, 92.61), iii) Syphicheck, 74.47% (63.94, 82.13), and iv) VisiTect, 74.26% (53.62, 83.68), while specificities were: i) Syphicheck, 99.58% (98.91, 99.96), ii) VisiTect, 99.43% (98.22, 99.98), iii) SD Bioline, 97.95%(92.54, 99.33), and iv) Determine, 95.85% (92.42, 97.74).

Conclusions

Rapid and POC treponemal tests reported sensitivity and specificity estimates comparable to laboratory-based treponemal tests. In resource limited settings, where access to screening is limited and where risk of patients lost to follow up is high, the introduction of these tests has already been shown to improve access to screening and treatment to prevent stillbirths and neonatal mortality due to congenital syphilis. Based on the evidence, it is concluded that rapid and POC tests are useful in resource limited settings with poor access to laboratories or screening for syphilis.     

Plasminogen-Based Capture Combined with Amplification Technology for the Detection of PrPTSE in the Pre-Clinical Phase of Infection

Background

Variant Creutzfeldt-Jakob disease (vCJD) is a neurodegenerative infectious disorder, characterized by a prominent accumulation of pathological isoforms of the prion protein (PrP^TSE) in the brain and lymphoid tissues. Since the publication in the United Kingdom of four apparent vCJD cases following transfusion of red blood cells and one apparent case following treatment with factor VIII, the presence of vCJD infectivity in the blood seems highly probable. For effective blood testing of vCJD individuals in the preclinical or clinical phase of infection, it is considered necessary that assays detect PrP^TSE concentrations in the femtomolar range.

Methodology/Principal Findings

We have developed a three-step assay that firstly captures PrP^TSE from infected blood using a plasminogen-coated magnetic-nanobead method prior to its serial amplification via protein misfolding cyclic amplification (PMCA) and specific PrP^TSE detection by western blot. We achieved a PrP^TSE capture yield of 95% from scrapie-infected material. We demonstrated the possibility of detecting PrP^TSE in white blood cells, in buffy coat and in plasma isolated from the blood of scrapie-infected sheep collected at the pre-clinical stage of the disease. The test also allowed the detection of PrP^TSE in human plasma spiked with a 10⁻⁸ dilution of vCJD-infected brain homogenate corresponding to the level of sensitivity (femtogram) required for the detection of the PrP^TSE in asymptomatic carriers. The 100% specificity of the test was revealed using a blinded panel comprising 96 human plasma samples.

Conclusion/Significance

We have developed a sensitive and specific amplification assay allowing the detection of PrP^TSE in the plasma and buffy coat fractions of blood collected at the pre-clinical phase of the disease. This assay represents a good candidate as a confirmatory assay for the presence of PrP^TSE in blood of patients displaying positivity in large scale screening tests.