Simultaneous determination of eight beta-blockers by gradient high-performance liquid chromatography with combined ultraviolet and fluorescence detection in corneal permeability studies in vitro

A gradient HPLC method with combined ultraviolet and fluorescence detection was developed for the simultaneous determination of eight beta-blockers (alprenolol, atenolol, metoprolol, nadolol, pindolol, propranolol, sotalol and timolol) in corneal permeability studies in vitro. Fluorescence detection with excitation wavelength at 230 nm and emission at 302 nm was selective for six of the compounds, whereas UV detection at 205 nm was able to detect all the compounds. Calibration was performed with fluorescence detection for six compounds from 50 or 200 nM to 3 microM, and with UV detection for all the eight compounds from 100 or 200 nM to 30 microM. With optimized fluorescence detection, detection limits between 0.7 and 1.3 nM (0.035-0.065 pmol per 50 microl injection) were obtained for atenolol, metoprolol, nadolol and sotalol. A mixture of eight beta-blockers was used in cassette dosing permeability studies with a cultured corneal epithelium. The HPLC method revealed marked differences in the permeation between hydrophilic and lipophilic beta-blockers through the corneal epithelial cell culture model.     

Impact of protein binding on the analytical detectability and anticancer activity of thymoquinone

Thymoquinone (TQ), an active component of Nigella sativa L., is known to have anti-cancer and anti-inflammatory effects; however, no studies on its analytical detection in serum and its protein binding have been published. Using high performance liquid chromatography analysis, we show that the average recovery of TQ from serum is 2.5% at 10 μg/ml of TQ and 72% at 100 μg/ml. The low recovery of TQ from serum is due to its extensive binding to plasma proteins, as more than 99% of TQ was bound within 30 min of incubation. The binding of TQ to the major plasma proteins, bovine serum albumin (BSA) and alpha −1 acid glycoprotein (AGP), was studied and found to be 94.5 ± 1.7% for BSA and 99.1 ± 0.1% for AGP. Mass spectrometric analysis revealed that TQ was bound covalently to BSA, specifically on Cyst-34. Using WST-1 proliferation assay, we showed that BSA plays a protective role against TQ-induced cell death; pre-incubation with BSA prevented TQ from exerting its anti-proliferative effects against DLD-1 and HCT-116 human colon cancer cells. On the other hand, binding of TQ to AGP did not alter its anti-proliferative activity against both cell lines. When TQ was pre-incubated with AGP prior to the addition of BSA, the activity of TQ against DLD-1 was maintained, suggesting that AGP prevented the binding of TQ to BSA. This is the first time the covalent binding and inhibitory effect of BSA on TQ is documented. These data offer new grounds for TQ future pharmacokinetic analysis in vivo.     

Transcription factor snail1 expression and poor survival in pharyngeal squamous cell carcinoma

Snail1, a key regulator of epithelial-mesenchymal transition (EMT), plays an important role in tumour progression. Previous studies of snail1 have mainly focused on the epithelial tumour cells. The objective of this study was to evaluate the expression of snail1 protein in endothelial cells, stromal myofibroblasts and malignant epithelial cells of pharyngeal squamous cell carcinomas (PSCC), as well as its relation to clinicopathological features and survival. One hundred and ten tissue microarray samples were analyzed for snail1 expression using immunohistochemistry. In endothelial cells snail1 expression was observed in 51 (48%) of 107 cases and it predicted reduced disease specific survival (DSS) (p=0.009). In 49 (46%) tumour samples snail1 immunostaining was detected in stromal myofibroblasts and there was a tendency to poorer DSS in that group (p=0.067). Snail1 expression in endothelial cells and stromal myofibroblasts is also associated with hypopharyngeal tumours (p=0.01 and p=0.038 respectively), increasing T category (T3-4) (p=0.005, p=0.037 respectively) and poorer general condition of the patient (Karnofsky performance status score <70; p=0.029, p=0.039 respectively). Moreover endothelial expression correlated with advanced stage (III-IV) (p=0.005) and poorer differentiation (grade 2-3; p=0.012). In malignant epithelial cells snail1 immunostaining was detected in 75 of 110 cases (68%). Expression of the protein was more common in hypopharyngeal tumours (p=0.044). Snail1 positive tumours associated with a lower Karnofsky performance status score (p=0.039) and regional failure (p=0.042). Our findings indicate that snail1 protein expression in endothelial cells and to some extent also in tumour stromal myofibroblasts seems to be a predictor of poor survival in PSCC. The presence of snail1 protein in tumour microenvironment rather than in malignant epithelial tumour cells may induce tissue remodelling and tumour progression.     

CD44 aptamer mediated cargo delivery to lysosomes of retinal pigment epithelial cells to prevent age-related macular degeneration

Age related macular degeneration (AMD) is a progressive, neurodegenerative disorder that leads to the severe loss of central vision in elderlies. The health of retinal pigment epithelial (RPE) cells is critical for the onset of AMD. Chronic oxidative stress along with loss of lysosomal activity is a major cause for RPE cell death during AMD. Hence, development of a molecule for targeted lysosomal delivery of therapeutic protein/drugs in RPE cells is important to prevent RPE cell death during AMD. Using human RPE cell line (ARPE-19 cells) as a study model, we confirmed that hydrogen peroxide (H₂O₂) induced oxidative stress results in CD44 cell surface receptor overexpression in RPE cells; hence, an important target for specific delivery to RPE cells during oxidative stress. We also demonstrate that the known nucleic acid CD44 aptamer - conjugated with a fluorescent probe (FITC) - is delivered into the lysosomes of CD44 expressing ARPE-19 cells. Hence, as a proof of concept, we demonstrate that CD44 aptamer may be used for lysosomal delivery of cargo to RPE cells under oxidative stress, similar to AMD condition. Since oxidative stress may induce wet and dry AMD, both, along with proliferative vitreoretinopathy, CD44 aptamer may be applicable as a carrier for targeted lysosomal delivery of therapeutic cargoes in ocular diseases showing oxidative stress in RPE cells.     

The correlation of vectorcardiographic changes to blood lactate concentration during an exercise test

In this study, the correlations between blood lactate concentration (BLC), different vector electrocardiogram (VECG) parameters, ventilatory parameters and heart rate during exercise and recovery periods were investigated. The aim was to clarify the relationships between VECG parameters and different exercise intensity markers. Six (25–37 years old) nonathlete, healthy, male participants took part in the study. All participants performed two different bicycle ergospirometric protocols (P1 and P2) in order to attain different lactate levels with different heart rate profiles. A principal component regression (PCR) approach is introduced for preprocessing the VECG components. PCR was compared to Sawitzcy Golay and wavelet filtering methods using simulated data. The performance of the PCR approach was clearly better in low signal-to-noise ratio (SNR) situations, and thus, it enables reliable VECG estimates even during intensive exercise. As a result, strong positive mean individual correlations between BLC and T-wave kurtosis (P1: r = 0.86 and P2: r = 0.8, p < 0.05 in 12/12 measurements) and negative correlation between BLC and cos RT (P1: r = ?0.7, P2: r = ?0.62, p < 0.05 in 8/12 measurements) were observed. The results of this study indicate that VECG parameters (in addition to heart rate) can make a significant contribution to monitoring of exercise intensity and recovery.     

Bile acid amidoalcohols: simple organogelators

Simple bile acid amide synthesis of lithocholic and deoxycholic acids with 2-aminoethanol and 3-aminopropanol are reported. The structural properties of these amides were examined by NMR spectroscopic, ESI-TOF mass spectral, and X-ray crystallographic methods. The gelation properties of these amides in common organic solvents and in three different water solutions were also investigated using Tyndall effect, SEM, TEM, and optical microscopy. 2-Hydroxyethylamides were found to be effective gelators in chlorinated organic solvents and 3-hydroxypropylamides in aromatic solvents. Both derivatives thicken neutral and acidic water solutions.     

Solution structure of the Ca2+-Binding EGF3-4 pair from vitamin K-dependent protein S: identification of an unusual fold in EGF3

Vitamin K-dependent protein S is a cofactor of activated protein C, a serine protease that regulates blood coagulation. Deficiency of protein S can cause venous thrombosis. Protein S has four EGF domains in tandem; domains 2-4 bind calcium with high affinity whereas domains 1-2 mediate interaction with activated protein C. We have now solved the solution structure of the EGF3-4 fragment of protein S. The linker between the two domains is similar to what has been observed in other calcium-binding EGF domains where it provides an extended conformation. Interestingly, a disagreement between NOE and RDC data revealed a conformational heterogeneity within EGF3 due to a hinge-like motion around Glu186 in the Cys-Glu-Cys sequence, the only point in the domain where flexibility is allowed. The dominant, bent conformation of EGF3 in the pair has no precedent among calcium-binding EGF domains. It is characterized by a change in the psi angle of Glu186 from 160 degrees +/- 40 degrees , as seen in ten other EGF domains, to approximately 0 degrees +/- 15 degrees . NOESY data suggest that Tyr193, a residue not conserved in other calcium-binding EGF domains (except in the homologue Gas6), induces the unique fold of EGF3. However, SAXS data, obtained on EGF1-4 and EGF2-4, showed a dominant, extended conformation in these fragments. This may be due to a counterproductive domain-domain interaction between EGF2 and EGF4 if EGF3 is in a bent conformation. We speculate that the ability of EGF3 to adopt different conformations may be of functional significance in protein-protein interactions involving protein S.     

Impact of Ten-Valent Pneumococcal Conjugate Vaccination on Invasive Pneumococcal Disease in Finnish Children – A Population-Based Study

Background

The ten-valent pneumococcal conjugate vaccine (PCV10) was introduced into the Finnish National Vaccination Program (NVP) in September 2010 with a 2+1 schedule (3, 5, 12 months) without catch-up vaccinations. We evaluated the direct and indirect effects of PCV10 on invasive pneumococcal disease (IPD) among children ≤5 years of age during the first three years after NVP introduction.

Methods

We conducted a population-based, observational follow-up study. The cohort of vaccine-eligible children (all children born June 1, 2010 or later) was followed from 3 months of age until the end of 2013. For the indirect effect, another cohort of older children ineligible for PCV10 vaccination was followed from 2011 through 2013. Both cohorts were compared with season- and age-matched reference cohorts before NVP introduction. National, population-based laboratory surveillance data were used to compare culture-confirmed serotype-specific IPD rates in the vaccine target and reference cohorts by using Poisson regression models.

Results

The overall IPD rate among vaccine-eligible children was reduced by 80% (95%CI 72 to 85); the reduction in vaccine-type IPD was 92% (95%CI 86 to 95). However, a non-significant increase in non-vaccine type IPD was observed. During 2012–2013, we also observed a 48% (95%CI 18 to 69) reduction in IPD among unvaccinated children 2 to 5 years of age, which was mostly attributable to the ten vaccine serotypes.

Conclusions

This is the first population-based study investigating the impact of PCV10 introduction without prior PCV7 use. A substantial decrease in IPD rates among vaccine-eligible children was observed. A smaller and temporally delayed reduction among older, unvaccinated children suggests that PCV10 also provides indirect protection against vaccine-type IPD. Changes in serotype distribution warrant continuous monitoring of potential increases in non-vaccine serotypes.     

Heartwood and sapwood development within maritime pine (<Emphasis Type="Italic">Pinus pinaster </Emphasis>Ait.) stems

Heartwood and sapwood development in maritime pine (Pinus pinaster Ait.) is reported based on 35 trees randomly sampled in four sites in Portugal. It was possible to model the number of heartwood rings with cambial age. The heartwood initiation age was estimated to be 13 years and the rate of sapwood transformation into heartwood was 0.5 and 0.7 rings year^–1 for ages below and above 55 years, respectively. Reconstruction of heartwood volume inside the tree stem was made by visual identification by image analysis in longitudinal boards along the sawn surfaces. This volume was integrated into the 3D models of logs and stems developed for this species representing the external shape and internal knots. Heartwood either follows the stem profile or shows a maximum value at 3.8 m in height, on average, while sapwood width is greater at the stem base and after 3 m remains almost constant up the stem. Up to 50% of tree height heartwood represents 17% of stem volume, in 83-year-old trees and 12–13% in 42 to 55-year-old trees. Tree variables such as stem diameter, DBH and tree total height were found to correlate significantly with the heartwood content.