Industrial hemp (Cannabis sativa L.)-a valuable alternative crop for growing in agricultural soils contaminated with heavy metals.

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.

Discovery of microRNA-like Small RNAs in Pathogenic Plant Fungus Verticillium nonalfalfae Using High-Throughput Sequencing and qPCR and RLM-RACE Validation.

Verticillium nonalfalfae (V. nonalfalfae) is one of the most problematic hop (Humulus lupulus 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 V. nonalfalfae 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 V. nonalfalfae 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 V. nonalfalfae 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 V. nonalfalfae pathotypes. Computational analysis predicted milRNA targets in the V. nonalfalfae 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.

Physiological and Biochemical Responses of Ungrafted and Grafted Bell Pepper Plants (Capsicum annuum L. var. grossum (L.) Sendtn.) Grown under Moderate Salt Stress.

The response of grafted bell pepper plants (Capsicum annuum L. var. grossum (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.

Risk of major bleeding in elderly patients with atrial fibrillation on direct oral anticoagulants: real world experience.

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.

Chitin-Binding Protein of Verticillium nonalfalfae Disguises Fungus from Plant Chitinases and Suppresses Chitin-Triggered Host Immunity.

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.

Comprehensive analysis of Verticillium nonalfalfae in silico secretome uncovers putative effector proteins expressed during hop invasion.

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.

Impact of Asymptomatic Pulmonary Embolism on the Long-Term Prognosis of Patients with Deep Venous Thrombosis.

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 (p = 0.676). Recurrent events were associated with unprovoked versus provoked DVT (hazard ratio [HR]: 5.01; 95% confidence interval [CI]: 2.25-11.17; p < 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; p < 0.001).

Identification of Novel Virulence-Associated Proteins Secreted to Xylem by Verticillium nonalfalfae During Colonization of Hop Plants.

Plant pathogens employ various secreted proteins to suppress host immunity for their successful host colonization. Identification and characterization of pathogen-secreted proteins can contribute to an understanding of the pathogenicity mechanism and help in disease control. We used proteomics to search for proteins secreted to xylem by the vascular pathogen Verticillium nonalfalfae during colonization of hop plants. Three highly abundant fungal proteins were identified: two enzymes, α-N-arabinofuranosidase (VnaAbf4.216) and peroxidase (VnaPRX1.1277), and one small secreted hypothetical protein (VnaSSP4.2). These are the first secreted proteins so far identified in xylem sap following infection with Verticillium spp. VnaPRX1.1277, classified as a heme-containing peroxidase from Class II, similar to other Verticillium spp. lignin-degrading peroxidases, and VnaSSP4.2, a 14-kDa cysteine-containing protein with unknown function and with a close homolog in related V. alfalfae strains, were further examined. The in planta expression of VnaPRX1.1277 and VnaSSP4.2 genes increased with the progression of colonization, implicating their role in fungal virulence. Indeed, V. nonalfalfae deletion mutants of both genes exhibited attenuated virulence on hop plants, which returned to the level of the wild-type pathogenicity in the knockout complementation lines, supporting VnaPRX1.1277 and VnaSSP4.2 as virulence factors required to promote V. nonalfalfae colonization of hop plants.

New educational approach for patients on warfarin improves knowledge and therapy control.

BACKGROUND: Patient education is essential for a safe and efficient oral anticoagulant treatment. We examined if a newly launched booklet with information on anticoagulant treatment with warfarin improved patient knowledge and the time spent in the therapeutic-range (TTR). METHODS: Standardized questionnaire was administered to 235 consecutive patients on warfarin to assess their baseline knowledge and readministered it 2 months after they received the booklet. A control group of 51 patients was interviewed only after the booklet had been distributed. RESULTS: Patient's knowledge at the baseline was unsatisfying (mean questionnaire score: 11/16) and a substantial progress was achieved after the educational intervention (mean questionnaire score: 13/16, p = 0.001). TTR is significantly increased after the intervention (63.4 ± 22.3 vs. 74.6 ± 23.8 %; p < 0.01). The mean questionnaire score and TTR after education were not different in the intervention and the control group. CONCLUSIONS: Knowledge about oral anticoagulant treatment and TTR is increased after the issue of the booklet in the majority of patients. However, for a small group of patients with unimproved knowledge new forms of education are needed.

Geomagnetic and strong static magnetic field effects on growth and chlorophyll a fluorescence in Lemna minor.

The geomagnetic field (GMF) varies over Earth's surface and changes over time, but it is generally not considered as a factor that could influence plant growth. The effects of reduced and enhanced GMFs and a strong static magnetic field on growth and chlorophyll a (Chl a) fluorescence of Lemna minor plants were investigated under controlled conditions. A standard 7 day test was conducted in extreme geomagnetic environments of 4 µT and 100 µT as well as in a strong static magnetic field environment of 150 mT. Specific growth rates as well as slow and fast Chl a fluorescence kinetics were measured after 7 days incubation. The results, compared to those of controls, showed that the reduced GMF significantly stimulated growth rate of the total frond area in the magnetically treated plants. However, the enhanced GMF pointed towards inhibition of growth rate in exposed plants in comparison to control, but the difference was not statistically significant. This trend was not observed in the case of treatments with strong static magnetic fields. Our measurements suggest that the efficiency of photosystem II is not affected by variations in GMF. In contrast, the strong static magnetic field seems to have the potential to increase initial Chl a fluorescence and energy dissipation in Lemna minor plants.

Unprovoked proximal venous thrombosis is associated with an increased risk of asymptomatic pulmonary embolism.

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.

Circadian rhythm of cholesterol synthesis in mouse liver: a statistical analysis of the post-squalene metabolites in wild-type and Crem-knock-out mice.

Circadian rhythms affect the total cholesterol levels in humans and animals, although their effect on cholesterol synthesis remain poorly understood. Here, we show for the first time that intermediates of the post-squalene portion of cholesterol synthesis also follow a circadian rhythm in the mouse liver. We used Crem-knock-out mice to investigate the effects of cAMP response element modulator (CREM) isoforms on cholesterol synthesis over time, as compared to wild-type mice. Multiple linear regression and cosinor statistical analysis were carried out on data obtained from 166 liver samples of mice, and the 24-h profiles were modelled across genotype, gender and zeitgeber time for lanosterol, 24,25-dihydrolanosterol, testis meiosis-activating sterol, and 7-dehydrocholesterol, along with cholesterol. The levels of these sterols were higher in female mice compared to males, although the genotype/gender factors showed no effects on the circadian oscillation of these sterols, except for 24,25-dihydrolanosterol. This study also highlights the importance of the statistical methods, where time, genotype and gender are the studied variables.

High D-dimer levels predict cardiovascular events in patients with chronic atrial fibrillation during oral anticoagulant therapy.

Atrial fibrillation (AF) is associated with hemostatic abnormalities and increased risk of thrombotic cardiovascular events even during oral anticoagulant therapy (OAT). The aim of our study was to evaluate the predictive value of hemostatic markers for the risk of major cardiovascular events during OAT. The study group comprised 113 patients with chronic AF (70.2 +/- 5.4 years old, 60% men), referred for OAT. Established clinical risk factors and levels of prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complexes (TAT), D-dimer, tissue plasminogen activator (t-PA) and plasminogen activator inhibitor 1 (PAI-1) antigen and activity, before and during OAT (after 3.9 +/- 0.7 months; INR 2.57 +/- 0.57) were determined. In all patients OAT significantly suppressed levels of F1+2 by 67%,TAT by 30% and D-dimer by 48% (all p <0.001). During an average follow-up of 44 months 22/111 (20%) patients suffered a combined cardiovascular event (stroke, myocardial infarction, peripheral vascular occlusion or vascular death). Patients with cardiovascular events were significantly older, had more frequent heart failure/systolic dysfunction and had significantly increased levels of D-dimer at entry (63 vs 39 ng/mL, p = 0.005) and during OAT (33 vs 18 ng/mL, p = 0.002), and of t-PA antigen at entry (14.3 vs 10.9 ng/mL, p = 0.02) and during OAT (15.0 vs 11.2 ng/mL, p = 0.05) (all values are medians). In multivariate Cox proportional hazard models, heart failure/systolic dysfunction (hazard ratio 2.91; 95% CI 1.17-7.26; p = 0.02), high levels of D-dimer on OAT (top vs. lower two quartiles) (hazard ratio 4.78, 95% CI 1.39-16.41; p = 0.01) and t-PA antigen levels (continuous variable) (hazard ratio 1.09; 95% CI 1.01-1.17; p = 0.02) were significantly associated with combined cardiovascular events. In conclusion, high levels of D-dimer and t-PA antigen during OAT are significant predictors of combined cardiovascular events in AF patients and, on this basis, could be useful additional markers of cardiovascular risk in such patients.