The Importance of Biotinylation for the Suitability of Cationic and Neutral Fourth-Generation Polyamidoamine Dendrimers as Targeted Drug Carriers in the Therapy of Glioma and Liver Cancer.

In this study, we hypothesized that biotinylated and/or glycidol-flanked fourth-generation polyamidoamine (PAMAM G4) dendrimers could be a tool for efficient drug transport into glioma and liver cancer cells. For this purpose, native PAMAM (G4) dendrimers, biotinylated (G4B), glycidylated (G4gl), and biotinylated and glycidylated (G4Bgl), were synthesized, and their cytotoxicity, uptake, and accumulation in vitro and in vivo were studied in relation to the transport mediated by the sodium-dependent multivitamin transporter (SMVT). The studies showed that the human temozolomide-resistant glioma cell line (U-118 MG) and hepatocellular carcinoma cell line (HepG2) indicated a higher amount of SMVT than human HaCaT keratinocytes (HaCaTs) used as a model of normal cells. The G4gl and G4Bgl dendrimers were highly biocompatible in vitro (they did not affect proliferation and mitochondrial activity) against HaCaT and U-118 MG glioma cells and in vivo (against Caenorhabditis elegans and Wistar rats). The studied compounds penetrated efficiently into all studied cell lines, but inconsistently with the uptake pattern observed for biotin and disproportionately for the level of SMVT. G4Bgl was taken up and accumulated after 48 h to the highest degree in glioma U-118 MG cells, where it was distributed in the whole cell area, including the nuclei. It did not induce resistance symptoms in glioma cells, unlike HepG2 cells. Based on studies on Wistar rats, there are indications that it can also penetrate the blood-brain barrier and act in the central nervous system area. Therefore, it might be a promising candidate for a carrier of therapeutic agents in glioma therapy. In turn, visualization with a confocal microscope showed that biotinylated G4B penetrated efficiently into the body of C. elegans, and it may be a useful vehicle for drugs used in anthelmintic therapy.

Repurposed Drugs Celecoxib and Fmoc-L-Leucine Alone and in Combination as Temozolomide-Resistant Antiglioma Agents-Comparative Studies on Normal and Immortalized Cell Lines, and on C. elegans.

Glioblastoma multiforme therapy remains a significant challenge since there is a lack of effective treatment for this cancer. As most of the examined gliomas express or overexpress cyclooxygenase-2 (COX-2) and peroxisome proliferator-activated receptors γ (PPARγ), we decided to use these proteins as therapeutic targets. Toxicity, antiproliferative, proapoptotic, and antimigratory activity of COX-2 inhibitor (celecoxib-CXB) and/or PPARγ agonist (Fmoc-L-Leucine-FL) was examined in vitro on temozolomide resistant U-118 MG glioma cell line and comparatively on BJ normal fibroblasts and immortalized HaCaT keratinocytes. The in vivo activity of both agents was studied on C. elegans nematode. Both drugs effectively destroyed U-118 MG glioma cells via antiproliferative, pro-apoptotic, and anti-migratory effects in a concentration range 50-100 µM. The mechanism of action of CXB and FL against glioma was COX-2 and PPARγ dependent and resulted in up-regulation of these factors. Unlike reports by other authors, we did not observe the expected synergistic or additive effect of both drugs. Comparative studies on normal BJ fibroblast cells and immortalized HaCaT keratinocytes showed that the tested drugs did not have a selective effect on glioma cells and their mechanism of action differs significantly from that observed in the case of glioma. HaCaTs did not react with concomitant changes in the expression of COX-2 and PPARγ and were resistant to FL. Safety tests of repurposing drugs used in cancer therapy tested on C. elegans nematode indicated that CXB, FL, or their mixture at a concentration of up to 100 µM had no significant effect on the entire nematode organism up to 4th day of incubation. After a 7-day treatment, CXB significantly shortened the lifespan of C. elegans at 25-400 µM concentration and body length at 50-400 µM concentration.

Exploring the Potential of Lapatinib, Fulvestrant, and Paclitaxel Conjugated with Glycidylated PAMAM G4 Dendrimers for Cancer and Parasite Treatment.

Fulvestrant (F), lapatinib (L), and paclitaxel (P) are hydrophobic, anticancer drugs used in the treatment of estrogen receptor (ER) and epidermal growth factor receptor (EGFR)-positive breast cancer. In this study, glycidylated PAMAM G4 dendrimers, substituted with F, L, and/or P and targeting tumor cells, were synthesized and characterized, and their antitumor activity against glioma U-118 MG and non-small cell lung cancer A549 cells was tested comparatively with human non-tumorogenic keratinocytes (HaCaT). All cell lines were ER+ and EGFR+. In addition, the described drugs were tested in the context of antinematode therapy on C. elegans. The results show that the water-soluble conjugates of G4P, G4F, G4L, and G4PFL actively entered the tested cells via endocytosis due to the positive zeta potential (between 13.57-40.29 mV) and the nanoparticle diameter of 99-138 nm. The conjugates of G4P and G4PFL at nanomolar concentrations were the most active, and the least active conjugate was G4F. The tested conjugates inhibited the proliferation of HaCaT and A549 cells; in glioma cells, cytotoxicity was associated mainly with cell damage (mitochondria and membrane transport). The toxicity of the conjugates was proportional to the number of drug residues attached, with the exception of G4L; its action was two- and eight-fold stronger against glioma and keratinocytes, respectively, than the equivalent of lapatinib alone. Unfortunately, non-cancer HaCaT cells were the most sensitive to the tested constructs, which forced a change in the approach to the use of ER and EGFR receptors as a goal in cancer therapy. In vivo studies on C. elegans have shown that all compounds, most notably G4PFL, may be potentially useful in anthelmintic therapy.

Fluorescence Studies of the Interplay between Metal-Enhanced Fluorescence and Graphene-Induced Quenching.

Fluorescence microscopy and spectroscopy were applied for studying the optical properties of a hybrid nanostructure, in which we combine plasmon-induced metal enhanced fluorescence with energy transfer to epitaxial graphene. Covering the layer of silver islands with a monolayer graphene, while turning on the efficient energy transfer from emitters, only moderately affects the enhancement of fluorescence attributed to the plasmon resonance in metallic nanostructures-as evidenced by the analysis of fluorescence decays. The results show that it is feasible to combine the properties of graphene with metal-enhanced fluorescence. The importance of the layer thickness of the emitters is also pointed out.

Spectral Selectivity of Plasmonic Interactions between Individual Up-Converting Nanocrystals and Spherical Gold Nanoparticles.

We experimentally demonstrate strong spectral selectivity of plasmonic interaction that occurs between α-NaYF4:Er3+/Yb3+ nanocrystals, which feature two emission bands, and spherical gold nanoparticles, with plasmon frequency resonant with one of the emission bands. Spatially-resolved luminescence intensity maps acquired for individual nanocrystals, together with microsecond luminescence lifetime images, show two qualitatively different effects that result from the coupling between plasmon excitations in metallic nanoparticles and emitting states of the nanocrystals. On the one hand, we observe nanocrystals, whose emission intensity is strongly enhanced for both resonant and non-resonant bands with respect to the plasmon resonance. Importantly, this increase is accompanied with shortening of luminescence decays times. In contrast, a significant number of nanocrystals exhibits almost complete quenching of the emission resonant with the plasmon resonance of gold nanoparticles. Theoretical analysis indicates that such an effect can occur for emitters placed at distances of about 5 nm from gold nanoparticles. While under these conditions, both transitions experience significant increases of the radiative emission rates due to the Purcell effect, the non-radiative energy transfer between resonant bands results in strong quenching, which in that situation nullifies the enhancement.

Comparison of Two Different Modes of Active Recovery on Muscles Performance after Fatiguing Exercise in Mountain Canoeist and Football Players.

BACKGROUND: The aim of this study is to assess if the application of different methods of active recovery (working the same or different muscle groups from those which were active during fatiguing exercise) results in significant differences in muscle performance and if the efficiency of the active recovery method is dependent upon the specific sport activity (training loads). DESIGN: A parallel group non-blinded trial with repeated measurements. METHODS: Thirteen mountain canoeists and twelve football players participated in this study. Measurements of the bioelectrical activity, torque, work and power of the vastus lateralis oblique, vastus medialis oblique, and rectus femoris muscles were performed during isokinetic tests at a velocity of 90°/s. RESULTS: Active legs recovery in both groups was effective in reducing fatigue from evaluated muscles, where a significant decrease in fatigue index was observed. The muscles peak torque, work and power parameters did not change significantly after both modes of active recovery, but in both groups significant decrease was seen after passive recovery. CONCLUSIONS: We suggest that 20 minutes of post-exercise active recovery involving the same muscles that were active during the fatiguing exercise is more effective in fatigue recovery than active exercise using the muscles that were not involved in the exercise. Active arm exercises were less effective in both groups which indicates a lack of a relationship between the different training regimens and the part of the body which is principally used during training.

Tension pneumothorax as a severe complication of endobronchial ultrasound-guided transbronchial fine needle aspiration of mediastinal lymph nodes.

This article presents a case report of a patient suffering from bullous emphysema and chronic obstructive pulmonary disease, who was diagnosed with tension pneumothorax after undergoing endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA). Tension pneumothorax is a severe but rare complication of EBUS-TBNA. It can result from lung injury caused by the biopsy needle or, in patients suffering from bullous emphysema, from spontaneous rupture of an emphysematous bulla resulting from increased pressure in the chest cavity during cough caused by bronchofiberoscope insertion. The authors emphasize that patients should be carefully monitored after the biopsy, and, in the case of complications, provided with treatment immediately in proper hospital conditions. Patients burdened with a high risk of complications should be identified before the procedure and monitored with extreme care after its completion.

Metal-enhanced fluorescence of chlorophylls in light-harvesting complexes coupled to silver nanowires.

We investigate metal-enhanced fluorescence of peridinin-chlorophyll protein coupled to silver nanowires using optical microscopy combined with spectrally and time-resolved fluorescence techniques. In particular we study two different sample geometries: first, in which the light-harvesting complexes are deposited onto silver nanowires, and second, where solution of both nanostructures are mixed prior deposition on a substrate. The results indicate that for the peridinin-chlorophyll complexes placed in the vicinity of the silver nanowires we observe higher intensities of fluorescence emission as compared to the reference sample, where no nanowires are present. Enhancement factors estimated for the sample where the light-harvesting complexes are mixed together with the silver nanowires prior deposition on a substrate are generally larger in comparison to the other geometry of a hybrid nanostructure. While fluorescence spectra are identical both in terms of overall shape and maximum wavelength for peridinin-chlorophyll-protein complexes both isolated and coupled to metallic nanostructures, we conclude that interaction with plasmon excitations in the latter remains neutral to the functionality of the biological system. Fluorescence transients measured for the PCP complexes coupled to the silver nanowires indicate shortening of the fluorescence lifetime pointing towards modifications of radiative rate due to plasmonic interactions. Our results can be applied for developing ways to plasmonically control the light-harvesting capability of photosynthetic complexes.

Prevalence of COPD and tobacco smoking in Malopolska region--results from the BOLD study in Poland.

INTRODUCTION: There is a paucity of population-based data on chronic obstructive pulmonary disease (COPD) prevalence in Poland. To address this problem we participated in the Burden of Obstructive Lung Disease (BOLD) Initiative which was developed to provide standardized methods for estimating the prevalence of COPD and its risk factors. OBJECTIVES: The study aimed to assess the prevalence of COPD and some of its risk factors in adults aged 40 years and older in the Malopolska region in southern Poland. PATIENTS AND METHODS: Region--representative sample was drawn, basing on the current census data. Detailed BOLD questionnaires as well as pre- and post-bronchodilator spirometry were applied to eligible individuals. RESULTS: Six hundred and three subjects provided questionnaire and spirometry data; of those 526 provided spirometry data of appropriate quality and were included in the final analysis. Estimated population prevalence of COPD was 22.1%, whereas 10.9% had COPD in GOLD Stage > or = 2. COPD was far more common in men and its prevalence increased with age and exposure to tobacco smoke, and was inversely related to education level. The prevalence of current tobacco smoking was 28% (34% and 22% in men and women, respectively). Seventy-nine percent of men and 42% of women were ever-smokers. Twenty-nine percent of never smoking individuals were passively exposed to tobacco smoke in their households. CONCLUSIONS: Our results confirm the high prevalence of COPD in the studied region of Poland and emphasize the need to increase efforts to improve COPD awareness and limit tobacco smoking habit.

The prevalence of alpha(1)-antitrypsin deficiency in a representative population sample from Poland.

AIM: Severe alpha(1)-antitrypsin (AAT) deficiency is one of the most common genetic disorders in Caucasians. The aim of the present study was to assess an unbiased frequencies of PI*S and PI*Z alleles using genotyping of a representative sample from the general population of Poland. METHODS: A random sample of age- and gender-stratified residents, aged 20 years or older, was drawn from the municipal directory of Kraków, Poland. The two most common deficiency alleles: PI*S and PI*Z were genotyped with qualitative real-time PCR using degenerative dual-labeled allele-specific fluorescent probes. RESULTS: In the total population of 859 adult subjects (mean age: 49.5 years; range: 20-90), 28 heterozygotes MS, 18 heterozygotes MZ and one homozygote S were diagnosed. The frequency of PI*S allele was 17.5 (95% CI: 11.6-23.9) per 1000; and that of PI*Z was 10.5 (95% CI: 5.8-15.7) per 1000. Therefore, the estimated prevalence of inherited severe AAT deficiency (homozygotes Z) in Poland is 1/9110 (95% CI: 1/4057-1/29,727). CONCLUSIONS: In the whole population of Poland comprising 38 millions, one may expect of about 4189 (95% CI: 1284-9406) subjects with severe AAT deficiency. These numbers are high enough to consider genetic testing being introduced into a common clinical practice.

Antibodies to N-homocysteinylated albumin as a marker for early-onset coronary artery disease in men.

N-homocysteinylated (Nepsilon-Hcy) proteins and corresponding antibodies have recently been discovered in humans and animals. Increased autoimmune response to Nepsilon-Hcy-proteins has been reported in stroke patients. The aim of the present study was to investigate whether antibodies against N-homocysteinylated albumin are associated with coronary artery disease (CAD). We studied 88 male patients aged 50 years or under with angiographically documented CAD and 100 age-matched apparently healthy men as controls. Serum levels of IgG antibodies against Nepsilon-Hcy-albumin were determined using an enzymelinked immunosorbent assay. Seropositivity to anti-Nepsilon-Hcy-albumin antibodies was 5-fold more frequent in CAD patients than in controls (52.3% vs 10.0%; p<0.0001). Plasma Hcy levels in CAD patients were also significantly higher in the former than in the latter group (medians, 13.0 microM vs 12.1 microM; p=0.026). Importantly, 41.2% of subjects with plasma total Hcy >14.5 mM were seropositive compared with 25.5% of normohomocysteinemic individuals (p=0.048). There was a weak correlation between anti-Nepsilon-Hcy-albumin antibodies and Hcy levels (r=0.16; p=0.03). By multivariate logistic regression analysis, seropositivity to anti-Nepsilon-Hcy-albumin antibodies was an independent predictor of early CAD (OR, 14.82; 95% CI, 4.47 to 49.19; p=0.00002). Interestingly, anti-Nepsilon-Hcy-albumin antibodies were associated with C-reactive protein levels (r=0.24; p=0.002). Seropositivity to anti-Nepsilon-Hcy-albumin antibodies showed no association with the MTHFR C677T polymorphism. Our results suggest that seropositivity to antibodies against Nepsilon-homocysteinylated albumin is associated with early-onset CAD. An autoimmune response to Nepsilon-Hcy-albumin may represent a novel mechanism involved in the early development of CAD.

Polymorphisms of the 5,10-methylenetetrahydrofolate and the methionine synthase reductase genes as independent risk factors for spina bifida.

We analyzed the role of the C677T polymorphism of the 5,10-methylenetetrahydrofolate and the A66G polymorphism of the methionine synthase reductase genes as risk factors for occurrence of spina bifida. The studied population included 106 mothers and 104 children from affected families, and a control group of 100 adults. We found statistically significant differences between the occurrence of the homozygosity in these polymorphisms in the groups of mothers and children with thoracolumbal defects (C677T polymorphism) and lumbosacral defects (A66G polymorphism). We postulate that these polymorphisms should be regarded as independent risk factors for spina bifida.