Loss of image quality in photobleaching during microscopic imaging of fluorescent probes bound to chromatin

Prolonged excitation of fluorescent probes leads eventually to loss of their capacity to emit light. A decrease in the number of detected photons reduces subsequently the resolving power of a fluorescence microscope. Adverse effects of fluorescence intensity loss on the quality of microscopic images of biological specimens have been recognized, but not determined quantitatively. We propose three human-independent methods of quality determination. These techniques require no reference images and are based on calculation of the actual resolution distance, information entropy, and signal-to-noise ratio (SNR). We apply the three measures to study the effect of photobleaching in cell nuclei stained with propidium iodide (PI) and chromomycin A3 (CA3) and imaged with fluorescence confocal microscopy. We conclude that the relative loss of image quality is smaller than the corresponding decrease in fluorescence intensity. Furthermore, the extent of quality loss is related to the optical properties of the imaging system and the noise characteristics of the detector. We discuss the importance of these findings for optimal registration and compression of biological images.     

A prospective randomised comparative study of endoscopic band ligation versus injection sclerotherapy of bleeding internal haemorrhoids in patients with liver cirrhosis

Background and study aimsBleeding internal haemorrhoids are common and used to be treated surgically with too many complications. Endoscopic therapy is trying to take the lead. Sclerotherapy and rubber band ligation are the candidates to replace surgical therapy especially in patients with liver cirrhosis. The aim of this study was to compare endoscopic injection sclerotherapy (EIS) to endoscopic rubber band ligation (EBL) regarding effectiveness and complications in the treatment of bleeding internal haemorrhoids in Egyptian patients with liver cirrhosis.Patients and methodsOne hundred and twenty adult patients with liver cirrhosis and bleeding internal haemorrhoids were randomised into two equal groups; the first treated with EBL using Saeed multiband ligator, and the second with EIS using either ethanolamine oleate 5% or N-butyl cyanoacrylate. All groups were matched as regards age, sex, Child score and pre-procedure Doppler values. Patients were followed up clinically and with abdominal ultrasound/Doppler for 6 months. Endoscopic and endosonography/Doppler was done before and one month after the procedure. Pre and post-procedure data were recorded and analysed.ResultsBoth techniques were highly effective in the control of bleeding from internal haemorrhoids with a low rebleeding [10% in the EBL group and 13.33% in the EIS group] and recurrence [20% in the EBL group 20% in the EIS group] rates. Child score had a positive correlation with rebleeding and recurrence in EIS group only.Pain score and need for analgesia were significantly higher while patient satisfaction was significantly lower in EIS compared to EBL [p < 0.05]. No significant difference between ethanolamine and cyanoacrylate subgroups was found [p > 0.05].ConclusionsBoth EBL and EIS were effective in the treatment of bleeding internal haemorrhoids in patients with liver cirrhosis. EBL had significantly less pain and higher patient satisfaction than EIS. EBL was also safer in patients with advanced cirrhosis.     

Validity and reliability of wheelchair sitting posture measures using Coach’s Eye in abled subjects

People in wheelchairs spend a long time in the sitting position and often incur alignment problems resulting in neck and back pain. This study: (1) assessed the validity/reliability of Coach’s Eye (CE) smart device application, (2) examined the effect of seat to back support angle adjustments on head, neck, and shoulder posture in the sitting position, and (3) compared changes in cervical rotation at each back support angle. Abled subjects sat in a wheelchair with back support angles positioned at 90°, 100°, and 110°. CE, as well as ImageJ software, was used to analyze three angles: sagittal head angle (SHA), cervical angle (CVA), and shoulder angle (SA). There were highly significant differences for CVA and SA (p < 0.001) among the three seat to back support angles. Validity of CE was examined by correlating CE with ImageJ scores. CE had high validity for all angles (r = 0.99, 0.98, 0.99 respectively, p < 0.001). Inter-rater reliability for SHA, CVA, and SA was high (intraclass correlation coefficient [ICC] ranged from 0.95 to 0.99). Head (CVA) and shoulder (SA) alignment was closest to neutral posture with back support angles set at 110° and 90°, respectively.     

Preparation and characterization of emamectin benzoate nanoformulations based on colloidal delivery systems and use in controlling Plutella xylostella (L.) (Lepidoptera: Plutellidae)

Colloidal delivery systems have been widely used as carriers for controlled delivery of pesticides to improve the efficacy and photostability of natural and semi-synthetic pesticides. In this study, we have synthesized emamectin benzoate nanoformulations (EB + NFs) depending on polymeric nanocapsules (PNC) and two types of the nanosilica, mesoporous nanosilica (MCM-48) and silicon dioxide nanoparticles (SNPs) as carriers for the emamectin benzoate (EB). The fabricated nanoformulations were characterized by using X-ray diffraction analysis, Fourier transform infrared spectroscopy, particle size, zeta potential, morphology, absolute recovery (AR), entrapment efficiency (EE), UV stability and release kinetics. The obtained results showed that the carriers had a remarkable loading ability for EB and improved the EB photostability. The EE% of nanoformulations were 92.84%, 87.45% and 71.19% for emamectin benzoate polymeric nanocapsules (EB + PNC), emamectin benzoate SNPs (EB + SNPs) and emamectin benzoate MCM-48 (EB + MCM-48) respectively. The insecticidal activity of EB + NFs against Plutella xylostella showed that the EB + SNPs was more effective than other EB + NFs and EB alone. The LC₅₀ values were 0.18, 4.03, 8.49 and 11.06 mg L⁻¹ for EB + SNPs, EB + MCM-48, EB + PNC and EB respectively. The obtained results suggest the colloidal delivery systems that used in this study could improve the efficacy and photostability for EB, and they are able to overcome the disadvantage of the natural and semi-synthetic pesticides such as environmental sensitivity and to increase the efficacy of pesticides, which eventually leads to reduce the dosage of pesticides needed, reducing the number of applications required in comparison to conventional formulations.

Colloidal delivery systems have been widely used as carriers for controlled delivery of pesticides to improve the efficacy and photostability of natural and semi-synthetic pesticides.     

Na+/H+ exchange in granulosa cells of the three largest preovulatory follicles of the domestic hen

Sodium-dependent intracellular pH (pHi) regulation was compared in granulosa cells from the three largest avian ovarian follicles by monitoring the pHi with biscarboxyethylcarboxyfluorescein, a dye whose fluorescence increases with alkalinity. Collagenase-dispersed granulosa cells obtained from the largest (F1), second largest (F2), and third largest (F3) preovulatory follicles about 2-3 hr prior to expected ovulation of F1 were used in the present study. The resting pHi measured in nominally bicarbonate free buffer with extra-cellular Na+ (Nao+ = 144 mM) and external pH (pHo) of 7.3 was about 6.8 in cells from F1, F2, and F3. There was no correlation between the stage of follicular development and the pHi whether the follicles were removed in the early or late preovulatory period. After acute cytoplasmic acidification by exposure of cells to nigericin in choline+ buffer, or by the abrupt removal of ammonium chloride, complete recovery of pHi occurred in 4-5 min. The rate and magnitude of the recovery were dependent upon the concentration of Nao+ and were abolished when Nao+ was replaced completely by choline+. Recovery in the presence of Nao+ was inhibited dose-dependently by amiloride (sodium-hydrogen exchange inhibitor). There was no difference between the rate and the extent of pHi recovery in acid-loaded cells obtained from F1, F2, and F3. Furthermore, by varying the concentration of Nao+ between 0 and 144 mM both young and matured granulosa cells extruded acid at the same rate. In addition, amiloride inhibited the Nao+ dependency of pHi recovery to a similar degree in F1, F2, and F3 cells. Our observations demonstrate in avian granulosa cells the existence of a Nao+-dependent, amiloride-sensitive pHi regulatory system that is equally effective in cells obtained from the three largest yolk-filled follicles.     

Micellar formulations of Crizotinib and Dasatinib in the management of glioblastoma multiforme

Glioblastoma multiforme (GBM) defies the currently practiced management of radiotherapy, chemotherapy and surgery and hence, it is associated with a high fatality rate with a median survival of 14.6 months. In our previous work investigating different tyrosine kinase inhibitors (TKIs), we established that a combination of Crizotinib and Dasatinib exerted the most potent effect on different GBM cell lines. In this work, to improve targeted therapy at the site of the tumour and avoid systemic toxicity, we exploited the enhanced permeability and retention effect by designing micellar formulations of these two TKIs. Crizotinib and Dasatinib were successfully encapsulated in poly(styrene-co-maleic acid) (SMA) micelles which were then evaluated for their physicochemical characteristics, anti-proliferative effect, mode of cell death, efficacy in spheroid models, effect on cell signalling, antiangiogenic potential and in vivo anticancer activity. Our results showed that this combination had induced a potent anti-proliferative effect in four GBM cell lines grown as a monolayer and as a spheroid. The combination was also efficacious in in vitro models of angiogenesis and vascular mimicry. In vivo data showed the enhanced activity of the micellar TKIs compared to free drugs. In conclusion, we proved that micellar formulations of Crizotinib and Dasatinib carry promising in vitro and in vivo efficacy that warrant further investigation.     

In Situ Encapsulation of Nile Red or Doxorubicin during RAFT‐Mediated Emulsion Polymerization via Polymerization‐Induced Self‐Assembly for Biomedical Applications

Hydrophobic agents, a fluorescent dye (Nile red, NR) or an anticancer drug (doxorubicin, DOX), are encapsulated into poly((N‐[3‐(dimethylamino) propyl] methacrylamide)‐b‐poly (methyl methacrylate) (PDMAPMA‐b‐PMMA) nanoparticles (NPs) via one‐pot reversible addition‐fragmentation chain‐transfer (RAFT)‐mediated emulsion polymerization in water. The macroRAFT, PDMAPMA, is chain‐extended with the methyl methacrylate (MMA), with the hydrophobic agents soluble in MMA, resulting in loaded NPs, with either NR or DOX via polymerization‐induced self‐assembly (PISA). The NR‐loaded NPs are visualized by structured illumination microscopy (SIM), thus indicating the successful loading of the fluorescent dye into the PMMA core. The DOX‐loaded NPs exhibit a sustained release profile over 5 d, showing a small burst effect during the first 2 h, as compared with the free DOX. The DOX‐loaded NPs show higher cell toxicity than the free DOX in RAW 264.7 cell line. The results demonstrate the potential of using emulsion polymerization for synthesis of tailored and reproducible NPs encapsulating hydrophobic agents.