Plastid differentiation during microgametogenesis determines green plant regeneration in barley microspore culture

Developing plants from in vitro culture of microspores or immature pollen grains (androgenesis) is a highly genotype-dependent process whose effectiveness in cereals is significantly reduced by occurrence of albino regenerants. Here, we examined a hypothesis that the molecular differentiation of plastids in barley microspores prior to in vitro culture affects the genotype ability to regenerate green plants in culture. At the mid-to-late uninucleate (ML) stage, routinely used to initiate microspore culture, the expression of most genes involved in plastid transcription, translation and starch synthesis was significantly higher in microspores of barley cv. ‘Mercada’ producing 90% albino regenerants, than in cv. ‘Jersey’ that developed 90% green regenerants. The ML microspores of cv. ‘Mercada’ contained a large proportion of amyloplasts filled with starch, while in cv. ‘Jersey’ there were only proplastids. Using additional spring barley genotypes that differed in their ability to regenerate green plants we confirmed the correlation between plastid differentiation prior to culture and albino regeneration in culture. The expression of GBSSI gene (Granule-bound starch synthaseI) in early-mid (EM) microspores was a good marker of a genotype potential to produce green regenerants during androgenesis. Initiating culture from EM microspores that significantly improved regeneration of green plants may overcome the problem of albinism

ATR, a DNA damage signaling kinase, is involved in aluminum response in barley

Ataxia Telangiectasia and Rad-3-related protein (ATR) is a DNA damage signaling kinase required for the monitoring of DNA integrity. Together with ATM and SOG1, it is a key player in the transcriptional regulation of DNA damage response (DDR) genes in plants. In this study, we describe the role of ATR in the DDR pathway in barley and the function of the HvATR gene in response to DNA damages induced by aluminum toxicity. Aluminum is the third most abundant element in the Earth’s crust. It becomes highly phytotoxic in acidic soils, which comprise more than 50% of arable lands worldwide. At low pH, Al is known to be a genotoxic agent causing DNA damage and cell cycle arrest. We present barley mutants, hvatr.g and hvatr.i, developed by TILLING strategy. The hvatr.g mutant carries a G6054A missense mutation in the ATR gene, leading to the substitution of a highly conserved amino acid in the protein (G1015S). The hvatr.g mutant showed the impaired DDR pathway. It accumulated DNA damages in the nuclei of root meristem cells when grown in control conditions. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis revealed that 60% of mutant nuclei possessed DNA nicks and breaks, whereas in the wild type only 2% of the nuclei were TUNELpositive. The high frequency of DNA damages did not lead to the inhibition of the cell cycle progression, but the mutant showed an increased number of cells in the G2/M phase. In response to treatments with different Al doses, hvatr.g showed a high level of tolerance. The retention of root growth, which is the most evident symptom of Al toxicity, was not observed in the mutant, as it was in its parent variety. Furthermore, Al treatment increased the level of DNA damages, but did not affect the mitotic activity and the cell cycle profile in the hvatr.g mutant. A similar phenotype was observed for the hvatr.i mutant, carrying another missense mutation leading to G903E substitution in the HvATR protein. Our results demonstrate that the impaired mechanism of DNA damage response may lead to aluminum tolerance. They shed a new light on the role of the ATR-dependent DDR pathway in an agronomically important species

Creation of a TILLING population in barley after chemical mutagenesis with sodium azide and MNU

Since the development of the Targeting Induced Local Lesions in Genome (TILLING) strategy, it has been applied in both plants and animals in many studies. The creation of an appropriate population is the first and most crucial step of TILLING. The goal is to obtain a highly mutagenized population that allows many mutations in any gene of interest to be found. Therefore, an effective method of mutation induction should be developed. A high mutation density is associated with saving time, costs, and the labor required for the development of a TILLING platform. The proper handling of the mutated generations, the establishment of a seed bank, and the development of a DNA library are essential for creating a TILLING population. The database in which all of the data from the molecular and phenotypic analyses are collected is a very useful tool for maintaining such population. Once developed, a TILLING population can serve as a renewable resource of mutations for research that uses both forward and reverse genetic approaches. In this chapter, we describe the methods for the development and maintenance of a TILLING population in barley

The influence of paclitaxel on hydrolytic degradation in matrices obtained from aliphatic polyesters and polyester carbonates

Biodegradable polymers have become common materials used in pharmacy and medicine due to their properties such as mechanical strength, biocompatibility and non-toxic degradation products. Different compositions of copolymers and also their chain microstructure may have an effect on matrices degradation and thus on the drug release profile. In our study, we aimed at the influence of paclitaxel content on hydrolytic degradation process of terpolymeric matrices. Hydrolytic degradation of three kinds of matrices (with 5 or 10% of paclitaxel and drug free matrices) prepared from three types of terpolymers was performed in vitro at 37OC in phosphate buffer solution (PBS, pH 7,4). The 1H and 13C NMR spectra of terpolymers were recorded. Thermal properties were monitored by differential scanning calorimetry (DSC). Molecular weight dispersity (D) and molecular weight were determined using gel permeation chromatography (GPC). The surface morphology was studied by means of the scanning electron microscopy (SEM). The most significant degradation was observed in case of poly(L-lactide-co-glycolide-co-ε-caprolactone) 44:32:24. Weight loss and water uptake were similar in the event of the same type of matrices obtained from the two poly(L-lactide-co-glycolide-co-TMC). Decelerated paclitaxel release in case of matrices with 51:26:23 molar ratio was noticed and it can be connected with higher content of carbonate units. Knowledge of paclitaxel influence on hydrolytic degradation process may contribute to receive valuable information about its release mechanisms from biodegradable terpolymers

Functional properties of polyurethane ureteral stents with PLGA and papaverine hydrochloride coating

Despite the obvious benefits of using ureteral stents to drain the ureters, there is also a risk of complications from 80-90%. The presence of a foreign body in the human body causes disturbances in its proper functioning. It can lead to biofilm formation on the stent surface, which may favor the development of urinary tract infections or the formation of encrustation, as well as stent fragmentation, complicating its subsequent removal. In this work, the effect of the polymeric coating containing the active substance-papaverine hydrochloride on the functional properties of ureteral stents significant for clinical practice were assessed. Methods: The most commonly clinically used polyurethane ureteral Double-J stent was selected for the study. Using the dip-coating method, the surface of the stent was coated with a poly(D,L-lactide-glycolide) (PLGA) coating containing the papaverine hydrochloride (PAP). In particular, strength properties, retention strength of the stent ends, dynamic frictional force, and the fluoroscopic visibility of the stent during X-ray imaging were determined. Results: The analysis of the test results indicates the usefulness of a biodegradable polymer coating containing the active substance for the modification of the surface of polyurethane ureteral stents. The stents coated with PLGA+PAP coating compared to polyurethane stents are characterized by more favorable strength properties, the smaller value of the dynamic frictional force, without reducing the fluoroscopic visibility.Web of Science2214art. no. 770

Risk of left atrial appendage thrombus in patients with atrial fibrillation and chronic kidney disease

Background: Atrial fibrillation (AF) and chronic kidney disease (CKD) are associated with an increased risk of ischemic stroke. The aim of this study was to compare the clinical characteristics, the incidence of left atrial appendage (LAA) thrombus and its predictors, and spontaneous echo contrast (SEC) in a population of patients with AF depending on estimated glomerular filtration rate (eGFR) values. Methods: This study included 1962 patients who underwent transesophageal echocardiographic examination (TEE) prior to cardioversion or ablation in the years 2014–2018 in three cardiac centers. Results: More than a quarter of AF patients had decreased eGFR ( < 60 mL/min/1.73 m2) and were characterized as a high-risk population, with more comorbidities, higher thromboembolic and bleeding risk compared to those with normal renal function. Oral anticoagulation (OAC) was prescribed in 97% and 93% of patients with decreased and normal eGFR, respectively, with a higher prevalence of prescribed non-vitamin K antagonist oral anticoagulants (NOACs). The incidence of LAA thrombus (24%, 9% and 4%) and SEC (25%, 25% and 19%) increases simultaneously with a decrease in eGFR ( < 30, 30–59 and > 60 mL/min/1.73 m2, respectively). Among patients prescribed reduced doses of NOAC, those with decreased eGFR were more often observed with LAA thrombus (10% vs. 2.5%). Non-paroxysmal AF, heart failure and previous bleeding were predictors of LAA thrombus, irrespective of eGFR value. CKD was the predictor of LAA thrombus in all patients including those with non-paroxysmal AF, males, without diabetes, without hypertension and with CHA2DS2-VASc < 2. Conclusions: Despite OAC, patients with concomitant AF and CKD remain at high risk for LAA thrombus formation

HorTILLUS - a rich and renewable source of induced mutations for forward/reverse genetics and pre-breeding programs in barley (Hordeum vulgare L.)

TILLING (Targeting Induced Local Lesions IN Genomes) is a strategy used for functional analysis of genes that combines the classical mutagenesis and a rapid, high-throughput identification of mutations within a gene of interest. TILLING has been initially developed as a discovery platform for functional genomics, but soon it has become a valuable tool in development of desired alleles for crop breeding, alternative to transgenic approach. Here we present the HorTILLUS (Hordeum—TILLING—University of Silesia) population created for spring barley cultivar “Sebastian” after double-treatment of seeds with two chemical mutagens: sodium azide (NaN3) and N-methyl-N-nitrosourea (MNU). The population comprises more than 9,600 M2 plants from which DNA was isolated, seeds harvested, vacuum-packed, and deposited in seed bank. M3 progeny of 3,481 M2 individuals was grown in the field and phenotyped. The screening for mutations was performed for 32 genes related to different aspects of plant growth and development. For each gene fragment, 3,072–6,912 M2 plants were used for mutation identification using LI-COR sequencer. In total, 382 mutations were found in 182.2Mb screened. The average mutation density in the HorTILLUS, estimated as 1 mutation per 477 kb, is among the highest mutation densities reported for barley. The majority of mutations were G/C to A/T transitions, however about 8% transversions were also detected. Sixty-one percent of mutations found in coding regions were missense, 37.5% silent and 1.1% nonsense. In each gene, the missense mutations with a potential effect on protein function were identified. The HorTILLUS platformis the largest of the TILLING populations reported for barley and best characterized. The population proved to be a useful tool, both in functional genomic studies and in forward selection of barley mutants with required phenotypic changes. We are constantly renewing the HorTILLUS population, which makes it a permanent source of new mutations.We offer the usage of this valuable resource to the interested barley researchers on cooperative basis

Niewłaściwe przepisywanie zredukowanej dawki NOAC w praktyce klinicznej — wyniki Polskiego Rejestru Migotania Przedsionków (POL-AF) u hospitalizowanych pacjentów

Introduction. Prescribing non-vitamin K antagonist oral anticoagulants (NOACs) in a reduced or full dosage is important for providing patients with atrial fibrillation (AF) with efficacious and safe treatment. The study aimed to evaluate the administration frequency of reduced NOAC dosages against the guidelines and analysis of factors predisposing to such a choice in patients with AF included in the Polish Atrial Fibrillation (POL-AF) Registry. Material and methods. The study included 1003 patients with AF treated with reduced dosages of NOACs hospitalized in ten Polish cardiology centers from January to December 2019. The criteria for appropriately reduced NOAC dosages was a dosage reduction of individual NOAC from the clinical studies, which was the basis for their registration. Results. Among the 1003 patients who were treated with a reduced dosage of NOACs, inappropriately reduced dosages were observed in 242 patients (24.1%): in 120 patients (29.3%) treated with rivaroxaban, in 93 patients (33.8%) treated with apixaban and in 29 patients (9.1%) treated with dabigatran (p &lt; 0.0001). Independent predictors of the use of inappropriately reduced dosages of NOACs were heart failure (odds ratio [OR] 1.55, confidence interval [CI]: 1.08–2.22) and hospitalization due to cardiac implantable electronic device (CIED) implantations/reimplantations (OR 2.01, CI: 1.27–3.17). Factors diminishing the chances of using inappropriately reduced dosages of NOACs were age (OR 0.98, CI: 0.97–0.998), vascular disease (OR 0.29, CI: 0.21–0.40) and creatinine clearance (CrCl) &lt; 60 mL/min (OR 0.37, CI: 0.27–0.52). Conclusions. In the group of patients treated with a reduced dosage of NOAC, 24.1% of patients had an inappropriately reduced dosage prescription, most frequently the patients receiving apixaban and rivaroxaban. The factor predisposing to prescribing an inappropriately reduced dosage of NOAC was heart failure and hospitalization due to CIED implantation/reimplantation. Label adherence to NOAC dosage is important to improve clinical outcomes in AF patients, and further investigation is needed to assess the best dosage of NOACs in the AF population.Wstęp. Przepisywanie doustnych przeciwkrzepliwych leków niebędących antagonistami witaminy K (NOAC) w dawce zredukowanej lub pełnej jest istotne dla zapewnienia pacjentom z migotaniem przedsionków (AF) skutecznego i bezpiecznego leczenia. Celem badania było ocenienie częstości stosowania zredukowanych dawek NOAC w stosunku do wytycznych oraz analiza czynników predysponujących do takiego wyboru u pacjentów z AF zarejestrowanych w Polskim Rejestrze Migotania Przedsionków (POL-AF). Materiał i metody. Badanie obejmowało 1003 pacjentów z AF leczonych zredukowanymi dawkami NOAC, hospitalizowanych w 10 polskich ośrodkach kardiologicznych od stycznia do grudnia 2019 roku. Kryterium stosowania odpowiednio zredukowanych dawek NOAC była redukcja dawki indywidualnego leku NOAC na podstawie badań klinicznych, które były podstawą ich rejestracji. Wyniki. Spośród 1003 pacjentów leczonych zredukowanymi dawkami NOAC, nieodpowiednio zredukowane dawki zaobserwowano u 242 pacjentów (24,1%): u 120 pacjentów (29,3%) leczonych rywaroksabanem, u 93 pacjentów (33,8%) leczonych apiksabanem oraz u 29 pacjentów (9,1%) leczonych dabigatranem (p < 0,0001). Niezależnymi czynnikami predykcyjnymi stosowania nieodpowiednio zredukowanych dawek NOAC były: niewydolność serca (iloraz szans [OR] 1,55; przedział ufności [CI]: 1,08–2,22) oraz hospitalizacja związana z wszczepieniem/reimplantacją kardioelektronicznych urządzeń wszczepialnych (CIED) (OR 2,01; CI: 1,27–3,17). Czynnikiem zmniejszającym szanse na stosowanie nieodpowiednio zredukowanych dawek NOAC były: wiek (OR 0,98; CI: 0,97–0,998), choroba naczyniowa (OR 0,29; CI: 0,21–0,40) i klirens kreatyniny (CrCl) < 60 ml/min (OR 0,37; CI: 0,27–0,52). Wnioski. W grupie pacjentów leczonych zredukowaną dawką NOAC, 24,1% pacjentów miało nieodpowiednio przepisane dawki, najczęściej pacjenci otrzymujący apiksaban i rywaroksaban. Czynnikami predysponującymi do przepisywania nieodpowiednio zredukowanej dawki NOAC były niewydolność serca oraz hospitalizacja związana z wszczepieniem/reimplantacją CIED. Przestrzeganie zaleceń dotyczących dawek NOAC jest istotne dla poprawy wyników klinicznych u pacjentów z AF, konieczne jest również dalsze badanie w celu oceny optymalnej dawki NOAC w populacji z AF