Environmental and genealogical effects on DNA methylation in a widespread apomictic dandelion lineage

DNA methylation in plant genomes occurs in different sequences and genomic contexts that have very different properties. DNA methylation that occurs in CG (mCG) sequence context shows transgenerational stability and high epimutation rate, and can thus provide genealogical information at short time scales. However, due to meta-stability and because mCG variants may arise due to other factors than epimutation, such as environmental stress exposure, it is not clear how well mCG captures genealogical information at micro-evolutionary time scales. Here, we analysed DNA methylation variation between accessions from a geographically widespread, apomictic common dandelion (Taraxacum officinale) lineage when grown experimentally under different light conditions. Using a reduced-representation bisulphite sequencing approach, we show that the light treatment induced differentially methylated cytosines (DMCs) in all sequence contexts, with a bias towards transposable elements. Accession differences were associated mainly with DMCs in CG context. Hierarchical clustering of samples based on total mCG profiles revealed a perfect clustering of samples by accession identity, irrespective of light conditions. Using microsatellite information as a benchmark of genetic divergence within the clonal lineage, we show that genetic divergence between accessions correlates strongly with overall mCG profiles. However, our results suggest that environmental effects that do occur in CG context may produce a heritable signal that partly dilutes the genealogical signal. Our study shows that methylation information in plants can be used to reconstruct micro-evolutionary genealogy, providing a useful tool in systems that lack genetic variation such as clonal and vegetatively propagated plants.     

High-performance liquid chromatographic determination of sphinganine and sphingosine in serum and urine of subjects from an endemic nephropathy area in Croatia

Endemic nephropathy (EN) is a chronic renal disease present as an endemic in Brodska Posavina, Croatia. The aim of the study was to assess the possible role of fumonisins, i.e., mycotoxins produced by Fusarium moniliforme, as causative agents for EN. Fumonisins inhibit ceramide synthase, the enzyme of de novo synthesis of sphingolipids, which leads to an increase in the sphinganine/sphingosine ratio. In the present study, a modified method has been used for the determination of the sphinganine/sphingosine ratio in human serum and urine of healthy subjects and EN patients from the endemic area. Free sphingoid bases, sphinganine and sphingosine, were obtained by base hydrolysis. Afterwards, precolumn ortho-phthaldialdehyde derivatisation, HPLC separation and quantification by fluorescence detection were performed. The results thus obtained pointed to a sphingolipid metabolism impairment, which may have been induced by fumonisins or fumonisin-like mycotoxins. As statistically significant differences were recorded in the subjects not yet affected with EN, an impairment in the metabolism of sphingolipids might be considered as an early indicator of EN.     

Study of the thermal stability of D-amino acid oxidase from Trigonopsis variabilis reveals enzyme inactivation via multiple steps

The thermal stability of a highly purified preparation of D-amino acid oxidase from Trigonopsis variabilis (TvDAO), which does not show microheterogeneity due to the partial oxidation of Cys-108, was studied based on dependence of temperature (20–60°C) and protein concentration (5–100 µmol L^?1). The time courses of loss of enzyme activity in 100 mmol L^?1 potassium phosphate buffer, pH 8.0, are well described by a formal kinetic mechanism in which two parallel denaturation processes, partial thermal unfolding and dissociation of the FAD cofactor, combine to yield the overall inactivation rate. Estimates from global fitting of the data revealed that the first-order rate constant of the unfolding reaction (k_a) increased 10⁴-fold in response to an increase in temperature from 20 to 60°C. The rate constants of FAD release (k_b) and binding (k_?b) as well as the irreversible aggregation of the apo-enzyme (k_agg) were less sensitive to changes in temperature, their activation energy (E_a) being about 52 kJ mol^?1 in comparison with an E_a value of 185 kJ mol^?1 for k_a. The rate-determining step of TvDAO inactivation switched from FAD dissociation to unfolding at high temperatures. The model adequately described the effect of protein concentration on inactivation kinetics. Its predictions regarding the extent of FAD release and aggregation during thermal denaturation were confirmed by experiments. TvDAO is shown to contain two highly reactive cysteines per protein subunit whose modification with 5,5′-dithio-bis (2-nitrobenzoic acid) was accompanied by inactivation. Dithiothreitol (1 mmol L^?1) enhanced up to 10-fold the recovery of enzyme activity during ion exchange chromatography of technical-grade TvDAO. However, it did not stabilize TvDAO at all temperatures and protein concentrations, suggesting that deactivation of cysteines was not responsible for thermal denaturation.     

Integrated approach for production of recombinant acetylacetone dioxygenase from Acinetobacter johnsonii

The C–C bond-cleaving acetylacetone dioxygenase Dke1 (EC 1.13.11.50) is a Fe^2?+?-dependent enzyme from Acinetobacter johnsonii that activates oxygen to convert a range of β-dicarbonyl substrates into α-oxo-aldehyde and acid products. Previous methods of downstream processing yielded Dke1 with substoichiometric Fe^2?+? content. This paper reports the integration of enzyme production in E. coli and affinity chromatography to prepare recombinant Dke1 that is completely loaded with its metal cofactor. The specific activity of Dke1 in E. coli cell extracts could be increased up to 20-fold, compared to optimized enzyme production with the natural host. Introduction of an affinity-tag allowed the isolation of fully active Dke1 in a single purification step with high yield (70%). Mass spectrometric analysis revealed at the level of >80% sequence coverage that the isolated enzyme corresponded exactly to the predicted gene product. Tagged Dke1 is shown to have retained the functional properties of native Dke1.     

Comparison between carotid stenting and carotid endarterectomy in early outcome

Carotid artery stenting (CAS) is a widely used method in prevention of stroke for carotid artery stenosis as an alternative to surgical treatment. Initial studies reveal higher morbidity and mortality rates for CAS than acceptable standards for carotid endarterectomy (CEA). The aim of this study was to compare results in a series of CAS with concurrent risk-matched group of CEA patients. The study included two groups of 50 patients with internal carotid artery stenosis. We compared early outcome (30 days after procedure) in risk-matched groups of patients that underwent these procedures. Post procedural complications were equally frequent in both groups. There was no significant difference in perioperative complication rates (P = 0.871). Comparison of these two methods shows that CAS and CEA are competitive methods for treatment of carotid artery stenosis. Particularly in symptomatic patients with high risk for surgery CAS is alternative treatment.     

Medical and psychological parameters in overweight and obese persons seeking treatment

The aim of the study was to analyse psychological characteristics and medical parameters in obese and overweight to identify the possible psychosocial consequences of obesity that may occur along with the numerous medical problems associated with excess body weight. Analysis was made on 296 patients (103 males and 193 females, median age 50, range 16-81) divided in three groups, depending on their Body mass index (BMI). Group I included 41 patients with BMI ranging from 25 to 29.9, group II included 170 patients with BMI from 30 to 34.9, and group III 85 patients with BM > or =35. We compared medical (glucose, cholesterol, triglycerides, HDL-cholesterol, systolic and diastolic blood pressure, body fat percentage) and psychological parameters (anxiety, depression, pros and cons of losing weight, self efficacy and four stages of change) in the patients included in the study. Univariate analysis has shown statistically significant difference among obese and overweight patients in goal weight, systolic and diastolic blood pressure, body fat percentage, glucose and cholesterol serum level. People with higher BMI (>30) found more advantages (pros) over disadvantages (cons) of weight loss but the level of anxiety and depression did not differ significantly among those 3 groups of patients. The results have shown that overweight and obese people have serious medical problems. They also differ in some psychological characteristics which have to be taken into consideration. Therefore, approach to these patients should be multidisciplinary, including dietary care, physical activity, psychological and medical care.     

Inhibition of proteasome deubiquitinating activity as a new cancer therapy

Ubiquitin-tagged substrates are degraded by the 26S proteasome, which is a multisubunit complex comprising a proteolytic 20S core particle capped by 19S regulatory particles. The approval of bortezomib for the treatment of multiple myeloma validated the 20S core particle as an anticancer drug target. Here we describe the small molecule b-AP15 as a previously unidentified class of proteasome inhibitor that abrogates the deubiquitinating activity of the 19S regulatory particle. b-AP15 inhibited the activity of two 19S regulatory-particle-associated deubiquitinases, ubiquitin C-terminal hydrolase 5 (UCHL5) and ubiquitin-specific peptidase 14 (USP14), resulting in accumulation of polyubiquitin. b-AP15 induced tumor cell apoptosis that was insensitive to TP53 status and overexpression of the apoptosis inhibitor BCL2. We show that treatment with b-AP15 inhibited tumor progression in four different in vivo solid tumor models and inhibited organ infiltration in an acute myeloid leukemia model. Our results show that the deubiquitinating activity of the 19S regulatory particle is a new anticancer drug target.     

Type I interferon drives dendritic cell apoptosis via multiple BH3-only proteins following activation by PolyIC in vivo

Background

DC are activated by pathogen-associated molecular patterns (PAMPs), and this is pivotal for the induction of adaptive immune responses. Thereafter, the clearance of activated DC is crucial to prevent immune pathology. While PAMPs are of major interest for vaccine science due to their adjuvant potential, it is unclear whether and how PAMPs may affect DC viability. We aimed to elucidate the possible apoptotic mechanisms that control activated DC lifespan in response to PAMPs, particularly in vivo.

Methodology/Principal Findings

We report that polyinosinic:polycytidylic acid (PolyIC, synthetic analogue of dsRNA) induces dramatic apoptosis of mouse splenic conventional DC (cDC) in vivo, predominantly affecting the CD8α subset, as shown by flow cytometry-based analysis of splenic DC subsets. Importantly, while Bim deficiency conferred only minor protection, cDC depletion was prevented in mice lacking Bim plus one of three other BH3-only proteins, either Puma, Noxa or Bid. Furthermore, we show that Type I Interferon (IFN) is necessary and sufficient for DC death both in vitro and in vivo, and that TLR3 and MAVS co-operate in IFNß production in vivo to induce DC death in response to PolyIC.

Conclusions/Significance

These results demonstrate for the first time in vivo that apoptosis restricts DC lifespan following activation by PolyIC, particularly affecting the CD8α cDC subset. Such DC apoptosis is mediated by the overlapping action of pro-apoptotic BH3-only proteins, including but not solely involving Bim, and is driven by Type I IFN. While Type I IFNs are important anti-viral factors, CD8α cDC are major cross-presenting cells and critical inducers of CTL. We discuss such paradoxical finding on DC death with PolyIC/Type I IFN. These results could contribute to understand immunosuppression associated with chronic infection, and to the optimization of DC-based therapies and the clinical use of PAMPs and Type I IFNs.     

The prevalence and pulmonary consequences of anxiety and depressive disorders in patients with asthma

The aim of this study was to determine the prevalence of anxiety and depression symptoms in outpatients with treated asthma and to determine the influence of anxiety and depression symptoms on lung function and asthma symptoms. The study was conducted in the pulmonary clinic of the Department of Pulmonary Diseases, Osijek University Hospital Centre, on 200 outpatients with asthma, aged 18-50 years, of which there were 65.5% women and 35.5% men. Each patient underwent a clinical examination with an extensive anamnesis and lung auscultation. The lung function was tested by spirometry. Demographic data and data on general and socioeconomic characteristics were evaluated using a questionnaire created internally for the purposes of this research, psychological status was assessed by HAD questionnaire, and Q test was used as a measure of asthma control. Based on the HAD questionnaire, 44.5% of asthma patients met the criteria for anxiety, and 24.5% of asthma patients met the criteria for depression. There was no significant correlation between asthma symptoms and the degree of anxiety or depression, while the pulmonary function of asthma patients negatively correlated with the degree of anxiety and depression. Pulmonary function in asthma patients with symptoms of anxiety and depression was significantly poorer than in asthma patients without anxiety and/or depression symptoms. The results show that among asthma patients there are large number of those who have symptoms of anxiety and depression. Asthma patients with symptoms of anxiety and depression have poorer lung function than patients with only asthma symptoms, however there is no significant correlation between the lung function and symptoms of asthma. We have confirmed that patients with anxiety symptoms visit general practitioners or EMS significantly more when compared to patients with depression symptoms.