Formulation,Characterisation and Stabilisation of Buccal Films for Paediatric Drug Delivery of Omeprazole

This study aimed to develop films for potential delivery of omeprazole (OME) via the buccal mucosa of paediatric patients. Films were prepared using hydroxypropylmethylcellulose (HPMC), methylcellulose (MC), sodium alginate (SA), carrageenan (CA) and metolose (MET) with polyethylene glycol (PEG 400) as plasticiser, OME (model drug) and L-arg (stabiliser). Gels (1% w/w) were prepared at 40°C using water and ethanol with PEG 400 (0–1% w/w) and dried in an oven (40°C). Optimised formulations containing OME and L-arg (1:1, 1:2 and 1:3) were prepared to investigate the stabilisation of the drug. Tensile properties (Texture analysis, TA), physical form (differential scanning calorimetry, DSC; X-ray diffraction, XRD; thermogravimetric analysis, TGA) and surface topography (scanning electron microscopy, SEM) were investigated. Based on the TA results, SA and MET films were chosen for OME loading and stabilisation studies as they showed a good balance between flexibility and toughness. Plasticised MET films were uniform and smooth whilst unplasticised films demonstrated rough lumpy surfaces. SA films prepared from aqueous gels showed some lumps on the surface, whereas SA films prepared from ethanolic gels were smooth and uniform. Drug-loaded gels showed that OME was unstable and therefore required addition of L-arg. The DSC and XRD suggested molecular dispersion of drug within the polymeric matrix. Plasticised (0.5% w/w PEG 400) MET films prepared from ethanolic (20% v/v) gels and containing OME: L-arg 1:2 showed the most ideal characteristics (transparency, ease of peeling and flexibility) and was selected for further investigation.KEY WORDS: buccal drug delivery, omeprazole, oral films, paediatric, plasticiser     

Studies on a PMCA-like protein in the outer mantle epithelium of <Emphasis Type="Italic">Anodonta cygnea</Emphasis>: insights on calcium transcellular dynamics

Early studies on the outer mantle epithelium (OME) cells of the freshwater bivalve Anodonta cygnea (Linnaeus, 1758) revealed high ionic calcium concentrations by electrophysiological methods and subsequently a high tendency to reach an intracellular toxic condition. This toxicity could be neutralized by specific mechanisms in the cytosol of OME cells of A. cygnea. The present immunocytochemistry studies of OME cells by light and transmission electron microscopy (TEM) clearly showed a positive reaction of an antibody directed against the human plasma membrane Ca²⁺-ATPase 1 (PMCA-1) in the cytoplasm of OME cells. Also, western blot analysis of different fractions of OME cells with anti human PMCA-1 and C28R2 antibodies confirmed the presence of a PMCA-like protein with an unusual topographical localization and a molecular weight of only 70–80 kDa. These results lead us to speculate that this PMCA-like protein is distributed either in the plasma membrane or in the entire cytosol, where it eventually regulates intracellular calcium levels. Interestingly, the antibody reactions showed seasonal variations, being highest in OME samples prepared during summer when A. cygnea live under natural acidosis and absent in samples taken in winter conditions, which is in accordance with the seasonal variation of shell calcification rates. During winter, PMCA-1 antibody reaction was also detected in OME cells of animals kept only under experimentally induced acidosis conditions. Therefore, we assume that a functional role for this PMCA-like protein in the intracellular calcium regulation of OME cells during the mineralization of the shells of A. cygnea can be speculated.     

Combination of Digital Image Processing and Statistical Data Segmentation to Enhance SPR and SPRi Sensor Responses

The work reports the combination of basic digital image processing (DIP) techniques and statistical segmentation strategy (SDS) to improve surface plasmon resonance curve (SPRc) and SPR imaging (SPRi) sensors' performance. The SPR image is used for sensing and monitoring biological events in the so-called SPR imaging process. In the traditional SPR process based on the attenuated total reflection (ATR) method, the image is used to create the SPR curve, and the curve features tracking is employed on sensing applications. The SPR curve features are enhanced after the pixels of the SPR image have been processed with low-complexity filters in the spatial domain (brightness, contrast, threshold, and morphological). The bootstrap was used as a statistical processing approach, selecting lines and columns from the image that was less affected by imperfections and noises in the image detector, and consequently reducing the SPR sensor instrumentation disturbances. Experimental tests with reversible binding water-mixture were performed, and both image and statistical processing were reported. The combination of DIP and SDS approaches improves the extraction of the curve features, increasing the performance in terms of resonance position sensitivity to 81%.

     

兰索拉唑与奥美拉唑对胃溃疡患者氧化应激水平和内皮功能的影响

目的:探讨兰索拉唑(LAN)与奥美拉唑(OME)治疗胃溃疡的临床效果及对患者血浆氧化应激水平和血管内皮功能的影响。方法:选取我院2014年1月~2016年10月收治的138例胃溃疡患者,按照随机数字表法均分为LAN组和OME组,并选取我院同期69例健康体检者为对照组。对照组不用任何药物,LAN组予以LAN治疗,OME组给予OME治疗。记录比较LAN组和OME组的临床疗效、Hp根除率及胃镜疗效;对照组与LAN组、OME组两组治疗前后血浆氧化应激指标、血管内皮功能指标水平;并评价LAN组与OME组的用药安全性。结果:总疗程结束后,LAN组总有效率、Hp根除率分别为94.2%、89.9%,较OME组(91.3%、87.0%)相比差异无统计学意义(P0.05)。经4个疗程后,LAN组胃镜总有效率为95.7%,明显高于OME组的85.5%(P0.05)。与对照组相比,LAN组、OME组治疗前血浆MDA、ET-1水平均显著上升(P0.01),SOD、NO水平均显著降低(P0.01)。与本组治疗前对比,LAN组、OME组治疗后血浆MDA、SOD、NO及ET-1水平均显著改善(P0.01),且LAN组治疗后各血浆因子水平改善效果均显著优于OME组(P0.01)。结论:LAN治疗胃溃疡改善患者临床症状、根除Hp的效果及安全性与OME相当,但LAN更能有效降低机体的氧化应激水平,调节血管内皮功能,促进溃疡愈合。     

Feasibility of using olive mill effluent (OME) as a wetting agent during the cultivation of oyster mushroom, Pleurotus ostreatus, on wheat straw

In this study, cultivation of oyster mushroom, Pleurotus ostreatus, on wheat straw substrate containing tap water and olive mill effluent (OME) mixture containing varying volume of OME was studied in order to investigate the feasibility of using OME as an alternative wetting agent and OME's impact on some fundamental food quality characteristics of mushrooms. Time period for mycelial colonization, primordium initiation and first harvest were comparatively evaluated with the control group. It was shown that the use of OME and tap water mixture consisting of OME up to 25% volumetrically was possible for the purpose of commercial mushroom production. Experimental results obtained from substrate containing 25% OME mixture showed no statistically significant difference compared to control group. The negative effects of increasing volume of OME in the mixture were also indicated by bioefficiency, which was found to be 13.8% for substrates wetted with 100% OME, whereas bioefficiency was 53.6% for control group. Increasing volume of OME in the mixture resulted in deformation of fruit body shape, whereas no significant difference in food quality was observed due to the higher amount of OME. This work suggested that the use of OME up to 25% as moisturizer could be considered, especially for the locations having significant number of olive mills and mushroom producers, both as an environmentally friendly solution for the safe and ecological disposal of OME and a practical way for recovering OME's economic value thereby.     

Codigestion of olive oil mill wastewaters with manure, household waste or sewage sludge

Combined anaerobic digestion of oil mill effluent(OME) together with manure, household waste (HHW) orsewage sludge was investigated. In batch experimentsit was shown that OME could be degraded into biogaswhen codigested with manure. In codigestion with HHWor sewage sludge, OME dilution with water (1:5) wasrequired in order to degrade it. Using continuouslystirred lab-scale reactors it was shown thatcodigestion of OME with manure (50:50 and 75:25 OMEto manure ratios) was successful with a theoreticalOME utilization of 75% and with approx. 87%reduction of the lipids content in OME. An OMEutilization of approx. 55%, and lipid reduction of73% was reached in codigestion with HHW (50:50 and75:25 OME to HHW ratios). The results showed thatthe high buffering capacity contained in manure,together with the content of several essentialnutrients, make it possible to degrade OME withoutprevious dilution, without addition of externalalkalinity and without addition of external nitrogensource.     

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.     

A Genetic Screen in Myxococcus xanthus Identifies Mutants That Uncouple Outer Membrane Exchange from a Downstream Cellular Response

Upon physical contact with sibling cells, myxobacteria transiently fuse their outer membranes (OMs) and exchange OM proteins and lipids. From previous work, TraA and TraB were identified to be essential factors for OM exchange (OME) in donor and recipient cells. To define the genetic complexity of OME, we carried out a comprehensive forward genetic screen. The screen was based on the observation that Myxococcus xanthus nonmotile cells, by a Tra-dependent mechanism, block swarm expansion of motile cells when mixed. Thus, mutants defective in OME or a downstream responsive pathway were readily identified as escape flares from mixed inocula seeded on agar. This screen was surprisingly powerful, as we found >50 mutants defective in OME. Importantly, all of the mutations mapped to the traAB operon, suggesting that there may be few, if any, proteins besides TraA and TraB directly required for OME. We also found a second and phenotypically different class of mutants that exhibited wild-type OME but were defective in a responsive pathway. This pathway is postulated to control inner membrane homeostasis by covalently attaching amino acids to phospholipids. The identified proteins are homologous to the Staphylococcus aureus MprF protein, which is involved in membrane adaptation and antibiotic resistance. Interestingly, we also found that a small number of nonmotile cells were sufficient to block the swarming behavior of a large gliding-proficient population. This result suggests that an OME-derived signal could be amplified from a few nonmotile producers to act on many responder cells.     

Correlation between the morpho-cytohistochemistry of the outer mantle epithelium of Anodonta cygnea with seasonal variations and following pollutant exposure

Seasonal and experimental conditions induce morphological and cytochemical variations in the outer mantle epithelium (OME) of the freshwater bivalve Anodonta cygnea, probably influencing the shell calcification mechanism. In this study, OME samples were taken from untreated animals in autumn, winter, spring and summer as well as from animals exposed to divalent metals (cadmium, chromium, lead, copper and zinc) and pesticides (setoxidim and dimethoate) and observed by light microscopy. The present results showed that OME cells have larger cell volumes and increased amounts of secreted macromolecules during spring and summer than in autumn and winter. This correlates with higher shell calcification rates in spring and summer and lower shell calcification rates in autumn and winter. The experiments showed that incubation with pollutants for 8 months dramatically reduced the cellular volume so that the density of cytoplasmic material appeared higher that in the control samples. The pronounced changes in OME cells suggest a significant decrease in secretory activity following exposure to toxic agents and this has implications for the shell calcification process.     

The immunoregulatory and allergy-associated cytokines in the aetiology of the otitis media with effusion

Inflammation in the middle ear mucosa, which can be provoked by different primary factors such as bacterial and viral infection, local allergic reactions and reflux, is the crucial event in the pathogenesis of otitis media with effusion (OME). Unresolved acute inflammatory responses or defective immunoregulation of middle inflammation can promote chronic inflammatory processes and stimulate the chronic condition of OME. Cytokines are the central molecular regulators of middle ear inflammation and can switch the acute phase of inflammation in the chronic stage and induce molecular-pathological processes leading to the histopathological changes accompanying OME. In this review we present cytokines identified in otitis media, immunoregulatory [interleukin (IL)-2, IL-10, transforming growth factor-beta]) and allergy associated (IL-4, IL-5, granulocyte-macrophage colony-stimulating factor), as crucial molecular regulators, responsible for chronic inflammation in the middle ear and the chronic condition of OME.     

Automated analysis of the cytokinesis-block micronucleus assay for radiation biodosimetry using imaging flow cytometry

The cytokinesis-block micronucleus (CBMN) assay is employed in biological dosimetry to determine the dose of radiation to an exposed individual from the frequency of micronuclei (MN) in binucleated lymphocyte cells. The method has been partially automated for the use in mass casualty events, but it would be advantageous to further automate the method for increased throughput. Recently, automated image analysis has been successfully applied to the traditional, slide-scoring-based method of the CBMN assay. However, with the development of new technologies such as the imaging flow cytometer, it is now possible to adapt this microscope-based assay to an automated imaging flow cytometry method. The ImageStream^X is an imaging flow cytometer that has adequate sensitivity to quantify radiation doses larger than 1 Gy while adding the increased throughput of traditional flow cytometry. The protocol and analysis presented in this work adapts the CBMN assay for the use on the ImageStream^X. Ex vivo-irradiated whole blood samples cultured for CBMN were analyzed on the ImageStream^X, and preliminary results indicate that binucleated cells and MN can be identified, imaged and enumerated automatically by imaging flow cytometry. Details of the method development, gating strategy and the dose response curve generated are presented and indicate that adaptation of the CBMN assay for the use with imaging flow cytometry has potential for high-throughput analysis following a mass casualty radiological event.     

The Role of <Emphasis Type="SmallCaps">l</Emphasis>-arginine in Inclusion Complexes of Omeprazole with Cyclodextrins

In this study, we investigate how the effect of l-arginine (ARG) and cyclodextrins upon omeprazole (OME) stability and solubility. The effect of the presence of ARG on the apparent stability constants (K_1:1) of the inclusion complexes formed between OME and each cyclodextrin, β-cyclodextrin (βCD), and methyl-β-cyclodextrin (MβCD) is studied by phase solubility diagrams and nuclear magnetic resonance (NMR) spectroscopy. The interaction of OME with those cyclodextrins, in the presence of ARG, is characterized using NMR spectroscopy and molecular dynamics simulations. ARG significantly increases the drug solubility and complex stability, in comparison to inclusion complexes formed in its absence. The effect is more pronounced for the OME:βCD complex. ARG also contributes to a larger stability of OME when free in aqueous solution. The combination of ARG with cyclodextrins can represent an important tool to develop stable drug formulations.     

Flexible Integration of Laser Myringotomy and Ventilation Tube for Bilateral Otitis Media with Effusion: Analysis of Laser Tympanostomy versus Ventilation Tube

Background

The aim of this study was to evaluate the efficacy of laser myringotomy (LM) compared to ventilation tube (VT), and to assess the clinical success criteria of LM-assisted VT insertion as the flexible alternatives avoiding GA for the treatment of bilateral consistent otitis media with effusion (OME).

Methods and Findings

LM under topical anesthesia was followed by VT insertion in cooperative children using Acuspot® 712 CO2 laser micromanipulator attached microscope. Sixty children failed VT and bilateral laser tympanostomy was done (group LL), and 130 children tolerated VT on one side but LM on the other side (group LV). The efficacy of LM was compared to VT regarding recurrent effusion and reoperation as the outcome measure; firstly, by ear-to-ear matched pair analysis in LV, and secondly between LL vs. LV. Long-term outcome was compared to control group who received both VT under GA (group GAVT) regarding the outcome of additional VT and GA.

Results

The effectiveness of LM per ear was equivocal as 46.9% and 40.8% in LV and LL respectively; but the effectiveness per children was further lower in LL as 28.3%, which was a limitation of LM for bilateral OME. LL required reoperation in 71.7% mostly requiring impending GA in 51.7% within 4.7 months, thus was a controversial treatment. But LV required GA in 20.8% during the 27.2 months long-term follow-up, which was second set of VT and adenoidectomy that were also comparably required in GAVT control with multiple GA.

Conclusion

Standard GAVT was more recommended for bilateral OME than bilateral LM (LL) in our practice. But LM was selectively effective for some children, that combined approach with LM plus VT provided comparable period to outgrow OME as effectively as GAVT, when LM was supplemented with one VT side with recovered hearing.     

Bacterial etiology of otitis media with effusion; focusing on the high positivity of Alloiococcus otitidis

The etiology of otitis media with effusion (OME) is unclear. The bacterial analyses of middle ear effusion (MEE) in OME may reveal important information regarding its etiology. Alloiococcus otitidis, Heamophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis were investigated by using microbiologic culture and a multiplex PCR method in the middle ear fluid of 32 children (54 samples) with chronic OME. PCR yielded positive results in 18 (33.3%) middle ear effusions while culture resulted positive for 3 (5.6%). The PCR method detected A. otitidis in 10 (18.5%) specimens, H. influenzae in 7 (13%), M. catarrhalis in 4 (7.4%) and S. pneumoniae in 2 (3.7%) specimens. The multiplex PCR method enhances the detection rate significantly compared to that of the conventional culture method. A. otitidis is the most common detected pathogen in the MEE of the OME.     

Correlation between the morpho-cytohistochemistry of the outer mantle epithelium of Anodonta cygnea with seasonal variations and following pollutant exposure

Seasonal and experimental conditions induce morphological and cytochemical variations in the outer mantle epithelium (OME) of the freshwater bivalve Anodonta cygnea, probably influencing the shell calcification mechanism. In this study, OME samples were taken from untreated animals in autumn, winter, spring and summer as well as from animals exposed to divalent metals (cadmium, chromium, lead, copper and zinc) and pesticides (setoxidim and dimethoate) and observed by light microscopy. The present results showed that OME cells have larger cell volumes and increased amounts of secreted macromolecules during spring and summer than in autumn and winter. This correlates with higher shell calcification rates in spring and summer and lower shell calcification rates in autumn and winter. The experiments showed that incubation with pollutants for 8 months dramatically reduced the cellular volume so that the density of cytoplasmic material appeared higher that in the control samples. The pronounced changes in OME cells suggest a significant decrease in secretory activity following exposure to toxic agents and this has implications for the shell calcification process.     

The role of protein tyrosine kinases in CYP1A1 induction by omeprazole and thiabendazole in rat hepatocytes

Benzimidazoles compounds like omeprazole (OME) and thiabendazole (TBZ) mediate CYP1A1 induction differently from classical aryl hydrocarbon receptor (AhR) ligands, 3-methylcholanthrene (3-MC) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). To clarify the involvement of an intracellular signal pathway in CYP1A1 induction by OME and TBZ, the TBZ, OME and 3-MC signal-transducing pathways were compared by using specific protein tyrosine kinase inhibitors in primary culture of rat hepatocytes. The effect of OME and TBZ (75-250 microM) on cytochrome P450 1A1 (CYP1A1) expression was therefore studied in primary cultures of rat hepatocytes after 24 h, 48 h and 72 h of exposure. Both compounds provoked a dose- and time-dependent increase in CYP1A1 (EROD activity, protein and mRNA levels), but OME was less effective at all the concentrations and times tested. The mechanism of benzimidazole-mediated induction of CYP1A1 was investigated by comparison with 3-MC, a prototypical AhR ligand. As expected, OME and TBZ were unable to displace [(3)H]-TCDD from its binding sites to the AhR in competitive binding studies. Moreover, classic tyrosine kinase inhibitor herbimycin A (HA) inhibited the two benzimidazoles-mediated CYP1A1 inductions, but only partially inhibited the 3-MC-mediated one. Another two tyrosine kinase inhibitors, Lavendustin A (LA) and genistein (GEN), had no effect on CYP1A1 induction by benzimidazoles and 3-MC. These results are consistent with the implication of a tyrosine kinase, most probably the Src tyrosine kinase, in the mechanism of CYP1A1 induction in rat hepatocytes.     

A novel principle for quantitation of fast intracellular calcium changes using Fura-2 and a modified image processing system--applications in studies of neutrophil motility and phagocytosis.

A new principle is described for imaging intracellular free calcium [Ca2+]i changes in single, living cells utilizing the fluorescent probe Fura-2. It is based upon video color mixing in real time and allows high-speed visualization, at maximum image resolution, of [Ca2+]i changes without digital image ratioing. The epifluorescence images produced by 340 and 380 nm excitations are stored in two memory buffers of a personal computer-based image processing system. Two video signals are generated independently from each buffer and connected to the red and green inputs of a video display. An image is this way created, in which [Ca2+]i shows up as a specific hue, whereas changes in dye concentration, light intensity, cell thickness show up as variations in brightness of the imaged cells. The method has advantages over conventional ratio imaging, notably simplicity and speed, since no calculations are made. Yet it can be combined with traditional digital image processing. The imaging technique allows monitoring of [Ca2+]i changes in rapidly moving cells, like neutrophils. It is demonstrated that during random locomotion on serum-coated glass surfaces, [Ca2+]i levels appeared to oscillate and that the frequency of the oscillations are related to locomotive activity. Furthermore, in Ca2+ free medium, the cells continue to move and phagocytose in the presence of Ca2+ ionophore (ionomycin) and 2 mM EGTA. In the presence of 1 mM extracellular Ca2+, ionomycin-treated cells were not able to move or phagocytose.     

Detection of soluble interleukin-2 receptor and soluble intercellular adhesion molecule-1 in the effusion of otitis media with effusion

We measured sIL-2R, TNF-alpha and sICAM-1 in the sera and middle ear effusions (MEEs) of patients with otitis media with effusion (OME). Although there was no signmcant difference between the sIL-2R levels of the serous and mucoid MEEs, they were significantly higher than serum sIL-2R levels of OME patients and healthy controls. TNF-alpha levels of the mucoid MEEs were significantly higher than those of the serous type. However, TNF-alpha was rarely detected in the sera of OME patients or healthy controls. We observed significant differences between the serous and mucoid MEEs with respect to their sICAM-1 levels, which were also higher than serum slCAM-1 levels of OME patients and healthy controls. Our findings suggested that IL-2, TNF-alpha and ICAM-1 could be significantly involved in the pathogenesis of OME through the cytokine network.     

超声弹性成像技术在乳腺肿块诊断中的研究进展及应用

超声弹性成像技术(UE)是一种新的超声成像技术,能够根据组织硬度进行成像,估计出组织内部的弹性信息,从而反映它的结构特点,该技术较传统触诊检查更加客观,在乳腺肿块的鉴别诊断中有较高的价值,其临床应用广泛并且得到了快速的发展。现就国内外文献对UE技术的原理、图像分析方法、技术研究进展及其在乳腺肿块鉴别诊断中的应用进行综述。     

Comparative analysis of iterative reconstruction algorithms with resolution recovery and new solid state cameras dedicated to myocardial perfusion imaging

New technologies are available in myocardial perfusion imaging. They include new software that recovers image resolution and limits image noise, multifocal collimators and dedicated cardiac cameras in which solid-state detectors are used and all available detectors are constrained to imaging just the cardiac field of view.These innovations resulted in shortened study times or reduced administered activity to patients, while preserving image quality.Many single center and some multicenter studies have been published during the introduction of these innovations in the clinical practice. Most of these studies were lead in the framework of “agreement studies” between different methods of clinical measurement. They aimed to demonstrate that these new software/hardware solutions allow the acquisition of images with reduced acquisition time or administered activity with comparable results (as for image quality, image interpretation, perfusion defect quantification, left ventricular volumes and ejection fraction) to the standard-time or standard-dose SPECT acquired with a conventional gamma camera and reconstructed with the traditional FBP method, considered as the gold standard.The purpose of this review is to provide the reader with a comprehensive understanding of the pro and cons of the different approaches summarizing the achievements reached so far and the issues that need further investigations.