Percolation limited emission intensity from upconverting NaYF4:Yb3+,Er3+ nanocrystals - a single nanocrystal optical study.

Upconverting nanocrystals (UCNC) have recently been subjected to intensive investigation due to their interesting optical properties and high potential for practical applications. Despite the level of attention paid to these materials, very low quantum yield is still an important issue. In order to break through this limitation, understanding of the emission intensity limitation is crucial. In this paper, we investigate the influence of percolation phenomena on the limitation of the emission intensity from NaYF4:Yb3+,Er3+ nanocrystals. We propose a numerical model and support this experimentally at the single nanocrystal level, explaining the influence of Yb3+ concentration on the optical properties of UCNC. Moreover, based on the experimental and numerical results, we explain the existence of the optimal Yb3+ concentration in the core architecture often reported in the literature. All the measurements have been performed using a custom-built wide-field fluorescence microscope to analyze the emission from hundreds of single nanocrystals and thus make analysis independent of UCNC concentration.

[Use of Porous Hydrogel as a 3D Scaffold for the Growth of Leukemic B Lymphocytes]. / Vyuzití porózního hydrogelu jako 3D scaffoldu pro rust leukemických B lymfocytu.

BACKGROUND: Primary human B cells chronic lymphocytic leukemia undergoes apoptosis, from which they can be rescued by contact with stromal cells or by the addition of specific soluble factor, when cultured in vitro. For research purposes of the behavior of CLL cells we created 3D in vitro model in which we simulated appropriate microenvironment for CLL cells to allow study the mechanism of survival of these cells in long-term cultivation. MATERIAL AND METHODS: Our aim was the scaffold structure to be geometrically similar to the 3D morphology of supporting bone marrow tissue in a trabecular bone; the 3D scaffold was also designed to conform to biocompatibility, sufficiently large surface area for cell attachment, high porosity for cell migration, proliferation and transport of nutrients. Another requirement was a partial transparency for inspection of cell model with optical techniques. We prepared 3D scaffolds from porous hydrogel poly (2-hydroxyethyl methacrylate) (pHEMA), poly (2-hydroxyethyl methacrylate-co-2-aminoethyl methacrylate) p (HEMA-co-AEMA) and p (HEMA-co-AEMA) modified with frequently used cell adhesion peptide Arg-Gly-Asp (RGD). All hydrogel scaffolds were manufactured in four pore diameters (125, 200, 300 and 350-450 µm). Scaffolds were tested with human bone marrow stromal cell line HS-5 and human embryonic kidney cell line HEK293. RESULTS: Hydrogel scaffold p (HEMA-co-AEMA) modified with adhesion peptide Arg-Gly-Asp (RGD) with pore diameter of 350-450 µm demonstrated that it is a convenient system for 3D cell cultivation, since it promotes interaction between the cells and also between the cells and the material. This scaffold was used for seeding of co-cultivation system of HS-5 cells with CLL-cells, which were stimulated through the CD40L signaling pathway as well as via the IL-4 pathway. Viability of B-CLL cells was higher in the presence of both stimulators than with each alone. CONCLUSIONS: We have shown that 3D scaffold technology is very useful for modeling of microsystems where the cancer cells behave like in their natural microenvironment.Key words: hematooncology - leukemia - hydrogel - stromal cells This work was supported by grant COST CZ LD15144 "Cellular and acellular grounds for regeneration of bones and teeth" awarded by the Ministry of Education, Youth and Sport of the Czech Republic. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 3. 2017Accepted: 26. 3. 2017.

Carbohydrate-modified magnetic nanoparticles for radical scavenging.

Maghemite (gamma-Fe2O3) nanoparticles, 12 nm in size, were prepared by co-precipitation of Fe(II) and Fe(III) chlorides with ammonium hydroxide and oxidation with hydrogen peroxide. To achieve stability and biocompatibility, obtained particles were coated with silica, to which glucose and ascorbic acid were bound by different mechanisms. The composite particles were thoroughly characterized by transmission electron microscopy, dynamic light scattering, elemental analysis, and FT-Raman and fluorescence spectroscopy to determine composition, morphology, size and its distribution, zeta-potential, and scavenging of peroxyl and hydroxyl radicals. As the particles showed promising antioxidative properties, they may have a possible application as a stable magnetically controlled scavenger of reactive oxygen species.

Silica-modified monodisperse hexagonal lanthanide nanocrystals: synthesis and biological properties.

Oleic acid-stabilized hexagonal NaYF4:Yb(3+)/Er(3+) nanocrystals, emitting green and red luminescence, were prepared by the high-temperature co-precipitation of lanthanide chlorides. By varying the reaction time and the Ln(3+)/Na(+) ratio, the nanocrystal size can be controlled within the range 16-270 nm. The maximum upconversion quantum yield is achieved under 970 nm excitation. The reverse microemulsion technique using hydrolysis and condensation of tetraethoxysilane is a suitable method to coat the nanocrystal surface with a silica shell to make the particles dispersible and colloidally stable in aqueous media. During the subsequent functionalization, (3-aminopropyl)trimethoxysilane introduced amino groups onto the silica to enable future bioconjugation with the target molecules. All specimens were characterized by TEM microscopy, electron and X-ray diffraction, ATR FT-IR spectroscopy, and upconversion luminescence. Finally, in vitro cytotoxicity and intracellular nanoparticle uptake (using confocal microscopy) were determined with human cervical carcinoma HeLa and mRoGFP HeLa cells, respectively. From the investigated particles, amino-functionalized NaYF4:Yb(3+)/Er(3+) nanocrystals internalized into the cells most efficiently. The nanoparticles proved to be nontoxic at moderate concentrations, which is important when considering their prospective application in biolabeling and luminescence imaging of various cell types.

Preparation of monodisperse porous silica particles using poly(glycidyl methacrylate) microspheres as a template.

Monodisperse macroporous poly(glycidyl methacrylate) (PGMA) microspheres were used as a template for preparing porous silica particles. The starting polymer microspheres that were 9.3 microm in size were synthesized by multistep swelling polymerization using a modified Ugelstad technique. Subsequently, silica (SiO2) was deposited on the surface and inside the PGMA microspheres to produce poly(glycidyl methacrylate)-silica hybrid particles (PGMA-SiO2). Upon calcination of the PGMA-SiO2 microspheres, porous silica particles were formed. The morphology, particle size, polydispersity and inner structure of the silica microspheres were investigated by scanning and transmission electron microscopy. Thermogravimetric analysis and dynamic adsorption of nitrogen determined the amount of silica formed and its specific surface area. Compared with the starting PGMA microspheres, the size of the porous silica particles decreased by up to 30%. These porous silica microspheres are promising for chromatography and biomedical applications.

Predictors of good clinical outcome in acute stroke patients treated with intravenous thrombolysis.

OBJECTIVES: Intravenous thrombolysis (IVT) is considered an effective treatment for acute ischemic stroke (IS). However, not all treated patients may achieve good outcome. The aim was to evaluate whether the initial NIHSS and DWI infarct volume could be the predictors for good outcome after IVT. PATIENTS AND METHODS: The set of 125 patients with consecutive hemispheric IS (78 men; mean age 66.0 ± 12.1 years) treated with IVT within 3 h was analyzed. DWI volume was measured on admission. Good outcome was defined as a score 0-2 in modified Rankin Scale. RESULTS: Multivariate logistic regression analysis showed initial NIHSS as an independent predictor of good outcome (P = 0.001). ROC curves showed baseline NIHSS ≤13.5 points and DWI volume ≤13.7 ml as cut-offs related to good outcome. CONCLUSIONS: The initial NIHSS and DWI volume might be the predictors for good clinical outcome in acute stroke patients treated with IVT. The initial NIHSS score seems to be more accurate.

Semisynthesis of C17:0 isoforms of sulphatide and glucosylceramide using immobilised sphingolipid ceramide N-deacylase for application in analytical mass spectrometry.

Sphingolipid ceramide N-deacylase (SCDase, EC 3.5.1.69) is a hydrolytic enzyme isolated from Pseudomonas sp. TK 4. In addition to its primary deacylation function, this enzyme is able to reacylate lyso-sphingolipids under specific conditions. We immobilised this enzyme on magnetic macroporous cellulose and used it to semisynthesise C17:0 glucosylceramide and C17:0 sulphatide, which are required internal standards for quantification of the corresponding glycosphingolipids (GSL) by tandem mass spectrometry. A high rate of conversion was achieved for both lipids (80% for C17:0 sulphatide and 90% for C17:0 glucosylceramide). In contrast to synthesis with a soluble form of the enzyme, use of immobilised SCDase significantly reduced the contamination of the sphingolipid products with other isoforms, so further purification was not necessary. Our method can be effectively used for the simple preparation of specifically labelled sphingolipids of high isoform purity for application in mass spectrometry.

[The importance of determining the viability of the myocardium prior to revascularisation in patients with ischaemic cardiomyopathy and left ventricular systolic dysfunction]. / Význam stanovení viability myokardu pred revaskularizací u pacientu s ischemickou kardiomyopatií a systolickou dysfunkcí levé komory srdecní.

Chronic heart failure is a worldwide clinical syndrome with substantial socio-economical impact. The aim of this overview is the summary of contemporary knowledge in epidemiology, elementary pathophysiological implications and some possibilities of treatment of ischemic cardiomyopathy, which represents the most frequent cause of heart failure in developed countries. Emphasis is on the right indication for myocardial revascularization as a causal option of therapy of ischemic cardiomyopathy, timing and risk of revascularization and especially on importance of assessment of viability of myocardium using presently available imaging methods.

[Preoperative assessment of myometrial invasion in endometrial cancer patients by ultrasonography and magnetic resonance imaging (MRI)]. / Predoperacní zhodnocení myometriální invaze u pacientek s karcinomem endometria pomocí ultrazvuku a magnetické rezonance.

OBJECTIVE: To compare the accuracy of transvaginal sonography and magnetic rezonance imaging (MRI) in determining the depth of myometrial invasion in proven cases of endometrial cancer. DESIGN: A prospective study. SETTING: Department of Obstetrics and Gynecology, Department of Medical Genetics and Fetal Medicine, Department of Radiology, University Hospital, Olomouc. METHODS: Fifty five patients diagnosed as having FIGO stage I endometrial carcinoma were evaluated preoperatively by transvaginal ultrasound; 44 cases of the same group were also evaluated by magnetic resonance imaging (MRI). The degree of invasion evaluated by transvaginal sonography and by MRI was compared to the pathological specimens. RESULTS: Transvaginal sonography was successful in evaluating myometrial invasion in 44 of 55 cases (accuracy 80%, sensitivity 80%, specificity 88%, positive predictive value 77%, negative predictive value 87%). Evaluation with MRI was accurate in 37 of 44 cases (accuracy 84%, sensitivity 84%, specificity 91%, positive predictive value 81%, negative predictive value 91%). CONCLUSION: Although MRI is superior to transvaginal sonography in evaluating myometrial invasion, it is expensive and time consuming, and would not be suitable as a screening test for depth of invasion. On the other hand, transvaginal sonography is relatively low-cost technique, which can be easily performed and repeated. However, it requires more operator experience than MRI in order to achieve high accuracy.

Isolation of microbial DNA by newly designed magnetic particles.

Carboxyl group-containing magnetic nonporous poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (P(HEMA-co-GMA)) microspheres and cobalt ferrite nanoparticles modified with alginic acid (natural carboxylic polysaccharide) were used for isolation of microbial DNA of lactic acid bacteria (LAB) from dairy products, lyophilised cell cultures, and bacterial colonies grown on hard media, and Trichophyton fungi DNA from lyophilised cells. DNA from the samples with lysed cells was reversibly adsorbed to the particles in the presence of high poly(ethylene glycol) (PEG 6000) and sodium chloride concentrations. The optimal final PEG and NaCl concentrations were 9.1 wt.% and 2.0 M, respectively. The adsorbed DNA was released from the particles in low ionic strength TE buffer. The quality of isolated DNA was checked by PCR amplification. Moreover, PCR amplicons were isolated on cobalt ferrite nanoparticles modified with alginic acid and checked by restriction analysis.

Poly(2-hydroxyethyl methacrylate) microspheres/liquid poly(dimethylsiloxane) composition for correction of small defects in face: histological evaluation in animal experiment.

Two kinds of composition based on commercial liquid poly(dimethylsiloxane) and laboratory-made poly(2-hydroxyethyl methacrylate) (PHEMA) microspheres of different size fractions (30-40 or 125-180 microm) were prepared. Tissue reaction on injection of the compositions, optimum microsphere size and morphology were investigated in the experiments on rats. The microspheres induced foreign body reaction characterized by an increased content of fibroblasts and mild infiltration of injection field by inflammatory cells. The 125-180 microm microspheres seemed to be well covered with poly(dimethylsiloxane) and more uniformly distributed in the tissue than the 30-40 microm ones. As a result, the extent of foreign body reaction induced by the former microspheres was somewhat lower than that induced by the latter. Moreover, time-dependent degradation of 30-40 microm PHEMA microspheres was more pronounced than that of 125-180 microm ones, which can affect duration of the aesthetic effect after prospective facioplasty. Results of histological investigations demonstrate a good prospect of the proposed composition for contour and bulk facioplasty of small soft tissue defects and skin wrinkles.

[Deferral of coronary intervention based on measurement of myocardial fractional flow reserve]. / Osud nemocných po odlození koronární intervence na základe merení frakcní prutokové rezervy myokardu.

BACKGROUND: Myocardial fractional flow reserve (FFR) is a useful method in assessment of functional significance of coronary stenosis. Deferral of intervention of angiographically intermediate lesion based on FFR measurement is safe in selected patient population as previously described. The aim of the study was to assess mid-term results after deferring coronary intervention of intermediate lesion in a non-selected patient population with no respect to the extent of coronary artery disease and to the results of stress tests if performed. METHODS: A coronary intervention of angiographically intermediate lesion (40 - 70% according to QCA) was deferred in a group of 50 consecutive patients (33 men, mean age 60.8 +/- 10.2 y.) on the basis of FFR > or = 0.75 (mean FFR 0.89 +/- 0.06). FFR was measured in 62 lesions (mean stenosis diameter 55 +/- 7%, left anterior descending 34 lesions, circumflex artery 13 lesions, right coronary artery 15 lesions). One-vessel disease was presented in 14 pts (28%), 36 pts (72%) presented with multivessel disease (two-vessel disease in 27 pts - 54% and three-vessel disease in 9 pts - 18%). Stress test was positive in 15 pts, in 1 pts. negative, and in 3 pts. non-diagnostic. All-cause mortality, cardiac mortality, non-fatal myocardial infarction (MI) and ischemia driven target vessel revascularization (TLR) were recorded during follow-up. Severity of angina pectoris (CCS classification) and a need for antianginal treatment (beta-blockers, nitrates, calcium channel blockers) at the baseline and at the end of clinical follow-up was recorded. RESULTS: Follow-up was completed in 49 patients (98%). Mean time of follow-up is 15.4 +/- 2 months (range 12 - 22 months, median 15 months), two patients died (4 %)--one from colon cancer, the other patient died from lung cancer, there was not any cardiac death recorded, two patients (4%) had target vessel revascularization. Estimated 22 months event-free (all-cause death, MI, TLR) survival was (mean +/- SEM) 86 +/- 7%. There was a significant difference in symptom severity--mean grade of angina pectoris at baseline was 1.8 +/- 1.3, at follow-up 1.1 +/- 1.0 (p < 0.05). There was not difference in use of antianginal drugs was same at baseline and at follow-up (1.7 +/- 0.8 vs. 1.7 +/- 0.7). Thirty-five patients (71%) were treated by statins. CONCLUSIONS: Deferring of coronary interventions of intermediate stenosis based on FFR measurement is safe in a mid-term follow-up. Despite of the same intensity of antianginal treatment there was a significant decrease in symptom severity.

[Use of magnetic resonance in preoperative evaluation of urinary tract obstruction in children--initial results]. / Vyuzití magnetické rezonance pri predoperacním vysetrení obstrukcí mocových cest u detí--první výsledky.

OBJECTIVE: To evaluate hitherto assembled experience with the application of magnetic resonance in the preoperative diagnosis of patients with obstruction of the upper urinary pathways. METHOD: Urography using magnetic resonance was performed in 22 children aged 1 month to 17 years with a sonographically assessed dilatation of the upper urinary pathways. The examinations were compared with concurrent excretory urography and formed part of a complex of preoperative examinations. RESULTS: In seven patients magnetic urography was evaluated as more accurate than excretory urography, in five instances excretory urography was more accurate, in the remaining children the results were comparable. CONCLUSION: The advantage of urography implemented by magnetic resonance is the short examination time, its non-invasive character, absence of ionizing radiation, impossible allergenization, and the examination does not depend on the functional fitness of the kidney. The results of examination are comparable with X-ray excretory urography.

[Angiographically marginally significant coronary stenoses--postponement of intervention based on measurement of myocardial fractional flow reserve]. / Angiograficky hranicne významné koronární stenózy--odlození intervence na základe merení frakcní prutokové rezervy myokardu.

INTRODUCTION: When evaluating angiographically marginal coronary stenoses (i.e. 40-70% reduction of the diameter of the arterial lumen) it is under certain conditions difficult to decide on their actual functional impact. Assessment of the fractional flow reserve (FFR) is a simple method based on assessment of intracoronary pressures during pharmacologically induced hyperaemia. For the severity of stenosis according to previous studies the liminal values is FFR lower than 0.75; furthermore it was proved that intervention of angiographically marginal stenoses with FFR values of 0.75 or more can be safely postponed. OBJECTIVE: Test the safety of FFR examination and take in a group of patients with marginally severe stenosis further steps according to results of FFR assessment. MATERIAL AND METHODS: During the period from January to Juky 2000 FFR assessments were made in a total of 34 patients (11 women, 23 men, mean age 62 +/- 12 years) who suffered from marginal stenosis of some coronary vessel. The FFR examination took place under pharmacologically induced hyperaemia after intracoronary adenosine administration. RESULTS: Measurements were made in a total of 41 stenoses. Only in two a value lower than 0.75 was found. In these patients coronary intervention was implemented; intervention was also made in two patients on account of technical problems and inconsistent results of FFR measurements. A FFR value of 0.75 or less was found in 37 stenoses (90%) and intervention was therefore postponed. Examination and the immediate subsequent course were without complications. CONCLUSION: According to initial experience FFR examination is a safe, simple and easily reproducible method. Based on the results of assessment and knowledge of the accomplished studies in the group of marginally significant stenoses in a great proportion of patients coronary intervention was postponed.

Enzymes immobilized on magnetic carriers: efficient and selective system for protein modification.

In order to obtain an economical, efficient and selective system for glycoprotein modification we prepared reactors with immobilized neuraminidase or (and) galactose oxidase. High storage and operational stability of the enzyme reactors was obtained by their immobilization through the carbohydrate parts of the enzyme molecules to hydrazide-modified supports. Magnetic and non-magnetic forms of bead cellulose and poly(HEMA-co-EDMA) microspheres were used for immobilization. These reactors can be used almost universally for the activation of ligands and for labelling of substances having a carbohydrate moiety.

Identification of poor prognostic features among patients requiring mechanical ventilation after hematopoietic stem cell transplantation.

Patients who develop respiratory failure requiring mechanical ventilation after hematopoietic stem cell transplantation (HSCT) have very high mortality. Several investigators have identified prognostic features that can be used to identify a subset of these patients who are virtually certain to die, yet these have never been prospectively assessed. The objectives of this study were to determine the accuracy of published prognostic features for mortality and to determine the survival of patients who recover from respiratory failure. A systematic review of the literature was undertaken to identify reported poor prognostic features and survival rates. The study validated the reported poor prognostic features on a prospective, multicenter inception cohort of 226 patients with respiratory failure requiring mechanical ventilation after HSCT. The main outcome measures were determination of a baseline probability of death, drawn from literature review; likelihood ratio of mortality for each prognostic feature determined from the validation cohort; conditional probability of death in the presence of each feature; and 6-month survival of those who recover. Patients requiring mechanical ventilation after HSCT have a baseline probability of death of 82% to 96%. In the setting of combined hepatic and renal dysfunction, the probability of death rises to 98% to 100%. Other previously reported prognostic features are less strongly associated with mortality. For patients who recover from respiratory failure, the proportion surviving 6 months or longer ranges from 27% to 88%. It was concluded that in patients requiring mechanical ventilation after HSCT, the presence of combined hepatic and renal dysfunction is highly predictive of death. The presence of this feature may justify the recommendation to withdraw life-sustaining measures.

Critical care of the hematopoietic stem cell patient.

Hematopoietic stem cell transplantation is evolving into a treatment modality with expanding indications and volume and with excellent outcomes, although it carries significant risk for morbidity and mortality affecting most major organ systems and often requires ICU care. With continuing improvements in supportive care and specific therapy of complications following HCT including the open-lung strategy of mechanical ventilation, use of nitric oxide, less toxic myeloablative regimens, newer classes of antibiotics, and improved immunosuppression strategies, it is hoped that mortality in this setting will continue to decline in coming years.

Properties of RNase A immobilized on magnetic Poly(2-hydroxyethyl methacrylate) microspheres.

Magnetic hydrogel microspheres 1.5 microm in size were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite, which formed the core of the particles. RNase A was coupled to the particles by the cyanuric chloride method. Gel electrophoresis of plasmid DNA pUC 19 (contaminated by bacterial RNA) confirmed RNA degradation with the immobilized enzyme. The effect of temperature and pH on the relative activity of immobilized RNase A was estimated after incubation of the samples at different temperatures (30-80 degrees C) and pH (4.0-8.0). Maximum relative activity was observed at 70 degrees C and pH 6.5. The matrices based on magnetic poly(HEMA) had a low tendency to adsorb RNA.

The short prodomain influences caspase-3 activation in HeLa cells.

Proteolytic activation of caspases is a key step in the process of apoptosis. According to their primary structure, caspases can be divided into a group with a long prodomain and a group with a short prodomain. Whereas long prodomains play a role in autocatalytic processing, little is known about the function of the short prodomain, for example the prodomain of caspase-3. We constructed caspase-3 variants lacking the prodomain and overexpressed these in HeLa and yeast cells. We found that removal of the caspase-3 prodomain resulted in spontaneous proteolytic activation of the protein when expressed in HeLa cells. This processing was only partially autocatalytic, as demonstrated by a catalytically inactive caspase-3 mutant. Co-expression of the anti-apoptotic protein XIAP (X-chromosome-linked inhibitor of apoptosis protein) completely blocked the observed spontaneous activation, which excluded a direct involvement of caspase-8. Our findings indicate that the short prodomain of caspase-3 serves as a silencing component in mammalian cells by retaining this executioner caspase in an inactive state.