During fungal infections, plant cells secrete chitinases, which digest chitin in the fungal cell walls. The recognition of released chitin oligomers via lysin motif (LysM)-containing immune host ...receptors results in the activation of defense signaling pathways. We report here that Verticillium nonalfalfae, a hemibiotrophic xylem-invading fungus, prevents these digestion and recognition processes by secreting a carbohydrate-binding motif 18 (CBM18)-chitin-binding protein, VnaChtBP, which is transcriptionally activated specifically during the parasitic life stages. VnaChtBP is encoded by the Vna8.213 gene, which is highly conserved within the species, suggesting high evolutionary stability and importance for the fungal lifestyle. In a pathogenicity assay, however, Vna8.213 knockout mutants exhibited wilting symptoms similar to the wild-type fungus, suggesting that Vna8.213 activity is functionally redundant during fungal infection of hop. In a binding assay, recombinant VnaChtBP bound chitin and chitin oligomers in vitro with submicromolar affinity and protected fungal hyphae from degradation by plant chitinases. Moreover, the chitin-triggered production of reactive oxygen species from hop suspension cells was abolished in the presence of VnaChtBP, indicating that VnaChtBP also acts as a suppressor of chitin-triggered immunity. Using a yeast-two-hybrid assay, circular dichroism, homology modeling, and molecular docking, we demonstrated that VnaChtBP forms dimers in the absence of ligands and that this interaction is stabilized by the binding of chitin hexamers with a similar preference in the two binding sites. Our data suggest that, in addition to chitin-binding LysM (CBM50) and Avr4 (CBM14) fungal effectors, structurally unrelated CBM18 effectors have convergently evolved to prevent hydrolysis of the fungal cell wall against plant chitinases and to interfere with chitin-triggered host immunity.
The vascular plant pathogen Verticillium nonalfalfae causes Verticillium wilt in several important crops. VnaSSP4.2 was recently discovered as a V. nonalfalfae virulence effector protein in the xylem ...sap of infected hop. Here, we expanded our search for candidate secreted effector proteins (CSEPs) in the V. nonalfalfae predicted secretome using a bioinformatic pipeline built on V. nonalfalfae genome data, RNA-Seq and proteomic studies of the interaction with hop. The secretome, rich in carbohydrate active enzymes, proteases, redox proteins and proteins involved in secondary metabolism, cellular processing and signaling, includes 263 CSEPs. Several homologs of known fungal effectors (LysM, NLPs, Hce2, Cerato-platanins, Cyanovirin-N lectins, hydrophobins and CFEM domain containing proteins) and avirulence determinants in the PHI database (Avr-Pita1 and MgSM1) were found. The majority of CSEPs were non-annotated and were narrowed down to 44 top priority candidates based on their likelihood of being effectors. These were examined by spatio-temporal gene expression profiling of infected hop. Among the highest in planta expressed CSEPs, five deletion mutants were tested in pathogenicity assays. A deletion mutant of VnaUn.279, a lethal pathotype specific gene with sequence similarity to SAM-dependent methyltransferase (LaeA), had lower infectivity and showed highly reduced virulence, but no changes in morphology, fungal growth or conidiation were observed. Several putative secreted effector proteins that probably contribute to V. nonalfalfae colonization of hop were identified in this study. Among them, LaeA gene homolog was found to act as a potential novel virulence effector of V. nonalfalfae. The combined results will serve for future characterization of V. nonalfalfae effectors, which will advance our understanding of Verticillium wilt disease.
(
) is one of the most problematic hop (
L.) pathogens, as the highly virulent fungal pathotypes cause severe annual yield losses due to infections of entire hop fields. In recent years, the RNA ...interference (RNAi) mechanism has become one of the main areas of focus in plant-fungal pathogen interaction studies and has been implicated as one of the major contributors to fungal pathogenicity. MicroRNA-like RNAs (milRNAs) have been identified in several important plant pathogenic fungi; however, to date, no milRNA has been reported in the
species. In the present study, using a high-throughput sequencing approach and extensive bioinformatics analysis, a total of 156 milRNA precursors were identified in the annotated
genome, and 27 of these milRNA precursors were selected as true milRNA candidates, with appropriate microRNA hairpin secondary structures. The stem-loop RT-qPCR assay was used for milRNA validation; a total of nine
milRNAs were detected, and their expression was confirmed. The milRNA expression patterns, determined by the absolute quantification approach, imply that milRNAs play an important role in the pathogenicity of highly virulent
pathotypes. Computational analysis predicted milRNA targets in the
genome and in the host hop transcriptome, and the activity of milRNA-mediated RNAi target cleavage was subsequently confirmed for two selected endogenous fungal target gene models using the 5' RLM-RACE approach.
Background
The efficacy and safety of direct oral anticoagulants is well established in patients with atrial fibrillation. However, data on their use in the oldest old patients (≥ 85 years), who have ...the highest risk of bleeding, is scarce.
Objective
The aim of this study was to evaluate the safety of direct oral anticoagulants in the oldest old patients with atrial fibrillation and assess the impact of age on major bleeding events.
Setting
Anticoagulation Clinic of the Department of Vascular Diseases, University Medical Centre Ljubljana, Slovenia.
Methods
From our single-centre prospective registry we enrolled 2260 consecutive atrial fibrillation patients aged ≥ 65 years that were started on dabigatran, rivaroxaban or apixaban. The mean duration of treatment exposure was 735 days. The primary outcome was the incidence of major bleeding. The incidence of thromboembolic events and death were also assessed. Potential risk factors for major bleeding were evaluated using Cox regression analysis.
Main outcome measure
Rate of major bleeding.
Results
During the follow-up 106 patients experienced major bleeding (2.3%/year). The oldest old patients (≥ 85 years) had the highest risk of any major bleeding (HR 2.50, 95% CI 1.44–4.32,
p
= 0.001), intracranial bleeding (HR 4.74, 95% CI 1.48–15.14,
p
< 0.01) and major gastrointestinal bleeding (HR 2.32, 95% CI 1.10–4.89,
p
< 0.03) compared to the group of patients aged 65–74 years, even though the majority of them were treated with reduced doses of direct oral anticoagulants. Significant predictors for major bleeding were age group ≥ 85 years (HR 2.52, 95% CI 1.43–4.47,
p
= 0.001) and history of bleeding (HR 3.32, 95% CI 1.87–5.90,
p
< 0.001). The incidence of a composite of stroke, transient ischemic attack and systemic embolism was 1.3%/year.
Conclusion
In this prospective real-world clinical study we have shown that the oldest old patients have the highest risk of major bleeding, which is further increased with a patient’s history of bleeding.
Hemp (
Cannabis sativa
L.) is a multiuse plant, which has been abundantly studied for phytoremediation purposes in recent years. The majority of experiments were performed in greenhouses with potted ...plants where hemp showed promising results. Only few studies tested hemp on site in heavy metal–polluted agricultural soil in real environmental conditions and practical assessments of hemp phytoremediation feasibility are lacking. We conducted a comprehensive study using 2 legal industrial hemp varieties (Futura 75 and Tisza) at three differently polluted locations (heavily polluted location, HP; moderately polluted location, MP; and slightly polluted location, SP) in the heavy metal contaminated Celje valley in Slovenia and determined the content of Pb, Zn, and Cd in 5 plant organs/tissues. The yield of each organ/tissue was determined as well to enable us to calculate the phytoremediation potential (PP). On average, plants grown in the HP location accumulated the highest values of all examined elements, followed by plants from the MP location and plants from the SP location, showing that the content of heavy metals in soil influences the accumulation in plants. Accumulation of Pb/Zn/Cd by plant organs/tissues was distributed in the following order: inflorescences (Pb-4.10/Zn-92.8/Cd-0.50 mg/kg) > seeds (Pb-1.79/Zn-92.6/Cd-0.27 mg/kg) > roots (Pb-1.15/Zn-15.0/Cd-0.44 mg/kg) > stem bark (Pb-0.42/Zn-12.4/Cd-0.23 mg/kg) > stem woody core (Pb-0.34/Zn-4.6/Cd-0.15 mg/kg). The only exception was for Cd, where roots accumulated a higher value than seed, yet lower than inflorescences. PP was calculated by multiplying hemp tissue/organ yield by the relative concentrations of heavy metal. The highest PP for Pb and Cd were achieved at the HP location (3.80 and 0.23 g/ha/vegetation period). On the other hand, tissue/organ yield was more important for high PP of Zn, where the SP location reached the highest PP for Zn (148.5 g/ha/vegetation period) due to the highest yields. Only seeds from HP and MP locations accumulated a too high content of Pb; otherwise, all other fibers and seeds can be safely used in the textile and food industry. Results of this study showed that hemp cannot be considered an efficient plant for the phytomanagement of contaminated areas. Nevertheless, hemp cultivation in heavy metal–polluted agricultural soils seems feasible since the majority of tissues/organs were not contaminated and different products can be obtained from various parts of the hemp plant.
Aims
Soils represent the natural habitat of entomopathogenic nematodes (EPNs). When moving in soil, EPNs are oriented to follow a chemical signal (chemotaxis).
Cannabis sativa
L. is known to secrete ...a very wide spectrum of secondary metabolites. Ethanol extracts (EE) of different
C. sativa
L. organs were used to study the effect on EPN chemotaxis.
Methods
The root, leaf and inflorescence EE of two hemp varieties and two medical cannabis breeding lines were used in laboratory chemotaxis assays with three EPN species (
Steinernema carpocapsae
,
S. feltiae
and
Heterorhabditis bacteriophora
) at 20 and 25 °C. The content of terpenes and cannabinoids in
C. sativa
L. inflorescences was measured as well.
Results
Overall, EPNs were most attracted or repelled by inflorescence extracts, followed by leaves and roots. The most abundant terpene in hemp inflorescences was trans caryophyllene (38.2% on average), the highest contents of total cannabidiol, total cannabigerol and total tetrahydrocannabinol were 9.65%, 0.89% and 0.62%, respectively.
Conclusions
The attraction effect of
S. sativa
L. EE is a highly interesting outcome and could lead to the development of attractant compounds in EPN biological control. Cannabinoids and terpenes may be responsible for pronounced effects on EPN chemotaxis.
Asymptomatic pulmonary embolism (PE) is present in at least one-third of patients with deep venous thrombosis (DVT). However, knowledge about its influence on the prognosis of patients is limited. ...The aim of this study was to assess the prognostic impact of asymptomatic PE in patients with DVT and to explore risk factors for recurrent venous thromboembolic events. A total of 200 consecutive patients with the first episode of objectively confirmed DVT without symptoms of PE were included. All patients underwent ventilation-perfusion scintigraphy within 48 hours of DVT confirmation. Patients with inconclusive scans further underwent computed tomography pulmonary angiography. At the time of inclusion and 4 weeks after discontinuation of anticoagulation, the levels of biomarkers of hemostasis and inflammation were assessed. Patients were followed up for a mean period of 4.2 ± 0.6 years. Recurrent episodes of venous thromboembolisms were recorded. Consistent with the literature, asymptomatic PE was present in 33.5% of the patients. During follow-up, 27 recurrent venous thromboembolisms were recorded, 20 presenting as DVT and 7 as symptomatic PE. Asymptomatic PE wasn't significantly associated with the rate of recurrence (
= 0.676). Recurrent events were associated with unprovoked versus provoked DVT (hazard ratio HR: 5.01; 95% confidence interval CI: 2.25-11.17;
< 0.001) and with increased versus normal D-dimer values, measured 4 weeks after discontinuation of anticoagulation (HR: 6.47; 95% CI: 2.96-14.17;
< 0.001).
Abstract Introduction Pulmonary embolism (PE) is common in patients with deep venous thrombosis (DVT). The outcome of DVT with concomitant symptomatic PE is worse than the outcome of isolated DVT. ...The risk factors for DVT and simultaneous asymptomatic PE have not been systematically studied yet. Aim To evaluate the frequency and risk factors for asymptomatic PE in patients with DVT. Patients/methods In 155 consecutive patients with a first episode of DVT and no PE symptoms, a ventilation-perfusion lung scan was performed. Body mass index (BMI) and waist-to-hip ratio (WHR) were calculated and concentrations of D-dimer, high-sensitivity CRP (hsCRP), tissue plasminogen activator (t-PA) and troponin were measured. Laboratory tests for thrombophilia were performed. Results Asymptomatic PE was present in 36% of patients. No differences in gender, age, BMI and WHR were found between the patients with and without PE. PE was more common in patients with proximal DVT than in those with distal DVT (42% vs. 17%, p < 0.01), and in patients with unprovoked DVT compared to patients with provoked DVT (51% vs. 28%, p < 0.01). The risk of silent PE was the highest in patients with unprovoked proximal DVT (OR, 6.9; 95% CI, 2.3–21.0). Patients with asymptomatic PE had significantly higher values of D-dimer, hsCRP, t-PA and troponin than patients with isolated DVT. Conclusions Asymptomatic PE affected more than one third of patients with a first DVT. Unprovoked proximal DVT is the most important risk factor for the occurrence of silent PE.
The response of grafted bell pepper plants (
L. var.
(L.) Sendtn.) to salt stress was investigated by analyzing the photosynthetic traits and mineral content of the plants and the metabolic ...composition of the fruit. The bell pepper variety "Vedrana" was grafted onto the salt-tolerant rootstock "Rocal F1" and grown at two salinities (20 mM and 40 mM NaCl) and control (0 mM NaCl) during the spring-summer period. On a physiological level, similar stomatal restriction of photosynthesis in grafted and ungrafted plants indicated that grafting did not alleviate water balance disturbances under increased salt exposure. Measurements of midday water potential did not show improved water status of grafted plants. The similar metabolic changes in grafted and ungrafted plants were also reflected in similarly reduced fruit yields. Thus, this grafting did not reduce the risk of ionic and osmotic imbalance in pepper plants grown under moderate salt treatment. Changes in the biochemical profiles of the pepper fruit were seen for both added-salt treatments. The fruit phenolic compounds were affected by rootstock mediation, although only for the July harvest, where total phenolics content increased with 40 mM NaCl treatment. Fruit ascorbic acid content increased with the duration of salt stress, without the mediation of the rootstock. The high salt dependence of this quality trait in pepper fruit appears to lead to more limited rootstock mediation effects.
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