Ultrasound Transmission Technique as a Potential Tool for Physical Evaluation of Monolithic Matrix Tablets

The aim of this study was to investigate the effects of tablet porosity and particle size fraction of compacted Starch acetate powders, with and without model drug caffeine, on acoustic properties of tablets. The ultrasound velocity was determined from the transmission measurements. Tablets of starch acetate (SA DS 2.7) powder with two particle size fractions of 0–53 and 0–710 μm were compressed with a compaction simulator. Porosities of tablets varied in the range from 12% to 43% for both particle size fractions. Strong associations were found between the ultrasound velocity and physical properties of the tablets such as porosity and particle size fraction. Interestingly, ultrasound velocity was practically insensitive to inclusion of the model drug caffeine with the concentrations used. Based on this study ultrasound transmission method is a potential non-destructive tool for studying structural changes of tablets and other solid dosage forms.     

Water-Level Fluctuations in Mediterranean Reservoirs: Setting a Dewatering Threshold as a Management Tool to Improve Water Quality

Water-level fluctuations, often linked to seasonal climatic trends, are a natural phenomenon which occur in almost all aquatic ecosystems. In some climatic regions, as the Mediterranean one, they are particularly wide due to the occurrence of two well separated periods: the rainy winter and the almost completely dry summer. Precipitation is concentrated in the first period, whereas in the second strong evaporation losses take place. According to these climatic features, and to ensure a continuous supply of water throughout the year, man-made lakes store water during winter and are subjected to dewatering during summer to compensate the lack of precipitation. These ecosystems are thus characterised by rather wide water level fluctuations which were observed to transform them from potentially warm monomictic lakes into polymictic or atelomictic ones. These changes deeply affect the biological structure and the functions of the water bodies impairing the response of some ecosystem properties, as resilience and resistance, since the impacts are immense enough to move the systems out of their homeostatic plateau of, respectively, deep or shallow lakes. In order to understand to what extent a reservoir can be “emptied” without changing its ecosystemic identity (deep or shallow lake sensu Padisák & Reynolds, 2003) and to set a “dewatering threshold”, the results from two different hydrological years, one with a dewatering so intense as to disrupt thermal stratification in midsummer, and the other one with water enough to allow the maintenance of the reservoir’s thermal structure throughout the summer, are compared. Former investigations have shown that the persistence of thermal stratification has a positive value in Sicilian reservoirs: a notable decrease in total phytoplankton biomass and in the relative occurrence of cyanoprokaryotes was observed in the high-level year with a stable thermal stratification. Although the solving of the external load problems causing eutrophication phenomena remain the main task to improve the water quality of this Mediterranean island, a management procedure, based on the maintaining of the ecosystem within its homeostatic plateau through the setting of a dewatering threshold, is suggested.     

Molecular Fingerprinting of Cyanobacteria from River Biofilms as a Water Quality Monitoring Tool

Benthic cyanobacterial communities from Guadarrama River (Spain) biofilms were examined using temperature gradient gel electrophoresis (TGGE), comparing the results with microscopic analyses of field-fixed samples and the genetic characterization of cultured isolates from the river. Changes in the structure and composition of cyanobacterial communities and their possible association with eutrophication in the river downstream were studied by examining complex TGGE patterns, band extraction, and subsequent sequencing of 16S rRNA gene fragments. Band profiles differed among sampling sites depending on differences in water quality. The results showed that TGGE band richness decreased in a downstream direction, and there was a clear clustering of phylotypes on the basis of their origins from different locations according to their ecological requirements. Multivariate analyses (cluster analysis and canonical correspondence analysis) corroborated these differences. Results were consistent with those obtained from microscopic observations of field-fixed samples. According to the phylogenetic analysis, morphotypes observed in natural samples were the most common phylotypes in the TGGE sequences. These phylotypes were closely related to Chamaesiphon, Aphanocapsa, Pleurocapsa, Cyanobium, Pseudanabaena, Phormidium, and Leptolyngbya. Differences in the populations in response to environmental variables, principally nutrient concentrations (dissolved inorganic nitrogen and soluble reactive phosphorus), were found. Some phylotypes were associated with low nutrient concentrations and high levels of dissolved oxygen, while other phylotypes were associated with eutrophic-hypertrophic conditions. These results support the view that once a community has been characterized and its genetic fingerprint obtained, this technique could be used for the purpose of monitoring rivers.     

C-Banding as a Simple Tool to Discriminate Between Micronuclei Induced by Clastogens and Aneugens

If mouse bone marrow preparations are treated with a classical C-banding procedure, it may be possible to distinguish between micronuclei with or without centromeres. This allows discrimination between micronuclei originating from chromosome breakage and those originating from chromosome loss. Thus, using C-banding, the micronucleus test can be used not only for the detection of clastogens but also aneugens. In this way, more exhaustive methods such as immunological staining using antikinetochore antibodies may not be necessary.     

Neural Differentiation of Mouse Neural Stem Cells as a Tool to Assess Developmental Neurotoxicity of Drinking Water in Taihu Lake

Biological Trace Element Research - The purpose of this study was to evaluate the protective effect of Du-Zhong cortex extract (DZCE) on lead acetate-induced bone loss in rats. Forty female...     

Protein-Bound Uremic Toxin Profiling as a Tool to Optimize Hemodialysis

Aim

We studied various hemodialysis strategies for the removal of protein-bound solutes, which are associated with cardiovascular damage.

Methods

This study included 10 patients on standard (3x4h/week) high-flux hemodialysis. Blood was collected at the dialyzer inlet and outlet at several time points during a midweek session. Total and free concentration of several protein-bound solutes was determined as well as urea concentration. Per solute, a two-compartment kinetic model was fitted to the measured concentrations, estimating plasmatic volume (V₁), total distribution volume (V_tot) and intercompartment clearance (K₂₁). This calibrated model was then used to calculate which hemodialysis strategy offers optimal removal. Our own in vivo data, with the strategy variables entered into the mathematical simulations, was then validated against independent data from two other clinical studies.

Results

Dialyzer clearance K, V₁ and V_tot correlated inversely with percentage of protein binding. All Ks were different from each other. Of all protein-bound solutes, K₂₁was 2.7–5.3 times lower than that of urea. Longer and/or more frequent dialysis that processed the same amount of blood per week as standard 3x4h dialysis at 300mL/min blood flow showed no difference in removal of strongly bound solutes. However, longer and/or more frequent dialysis strategies that processed more blood per week than standard dialysis were markedly more adequate. These conclusions were successfully validated.

Conclusion

When blood and dialysate flow per unit of time and type of hemodialyzer are kept the same, increasing the amount of processed blood per week by increasing frequency and/or duration of the sessions distinctly increases removal.     

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

Müller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling Müller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5 has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label Müller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.     

Optical Saturation as a Versatile Tool to Enhance Resolution in Confocal Microscopy

One of the most actively developing areas in fluorescence microscopy is the achievement of spatial resolution below Abbe's diffraction limit, which restricts the resolution to several hundreds of nanometers. Most of the approaches in use at this time require a complex optical setup, a difficult mathematical treatment, or usage of dyes with special photophysical properties. In this work, we present a new, to our knowledge, approach in confocal microscopy that enhances the resolution moderately but is both technically and computationally simple. As it is based on the saturation of the transition from the ground state to the first excited state, it is universally applicable with respect to the dye used. The idea of the method presented is based on a principle similar to that underlying saturation excitation microscopy, but instead of applying harmonically modulated excitation light, the fluorophores are excited by picosecond laser pulses at different intensities, resulting in different levels of saturation. We show that the method can be easily combined with the concept of triplet relaxation, which by tuning the dark periods between pulses helps to suppress the formation of a photolabile triplet state and effectively reduces photobleaching. We demonstrate our approach imaging GFP-labeled protein patches within the plasma membrane of yeast cells.     

Simulation as a New Tool to Establish Benchmark Outcome Measures in Obstetrics

Background

There are not enough clinical data from rare critical events to calculate statistics to decide if the management of actual events might be below what could reasonably be expected (i.e. was an outlier).

Objectives

In this project we used simulation to describe the distribution of management times as an approach to decide if the management of a simulated obstetrical crisis scenario could be considered an outlier.

Design

Twelve obstetrical teams managed 4 scenarios that were previously developed. Relevant outcome variables were defined by expert consensus. The distribution of the response times from the teams who performed the respective intervention was graphically displayed and median and quartiles calculated using rank order statistics.

Results

Only 7 of the 12 teams performed chest compressions during the arrest following the ‘cannot intubate/cannot ventilate’ scenario. All other outcome measures were performed by at least 11 of the 12 teams. Calculation of medians and quartiles with 95% CI was possible for all outcomes. Confidence intervals, given the small sample size, were large.

Conclusion

We demonstrated the use of simulation to calculate quantiles for management times of critical event. This approach could assist in deciding if a given performance could be considered normal and also point to aspects of care that seem to pose particular challenges as evidenced by a large number of teams not performing the expected maneuver. However sufficiently large sample sizes (i.e. from a national data base) will be required to calculate acceptable confidence intervals and to establish actual tolerance limits.     

Membrane Lipids, Selectively Diminished in Alzheimer Brains, Suggest Synapse Loss as a Primary Event in Early-Onset Form (Type I) and Demyelination in Late-Onset Form (Type II)

Abstract: Major membrane lipids were quantified in frontal (Brodmann area 9) and temporal (Brodmann areas 21 and 22) cortices, caudate nucleus, hippocampus, and frontal white matter of 12 cases with Alzheimer's disease (AD) type I (early onset), 21 cases with AD type II (late onset), and 20 age-matched controls. The concentration of gangliosides—a marker for axodendritic arborization—was reduced to 58–70% of the control concentration in all four gray areas (p < 0.0001) and to 81 % in frontal white matter (p < 0.01) of AD type I cases, whereas it was only significantly reduced in temporal cortex (p < 0.01), hippocampus (p < 0.05), and frontal white matter (p < 0.05) in AD type II cases. The concentration of phospholipids was also significantly reduced (p < 0.01–0.0001) in all four gray areas of AD type I cases but in no area of AD type II cases. The loss of cholesterol was only 50% of the corresponding phospholipid diminution in AD type I. These results suggested a pronounced loss of nerve endings in AD type 1. The characteristic membrane lipid disturbance in AD type II was a loss of myelin lipids. This is the first time a fundamental biochemical difference has been shown between the two major forms of AD.     

Formalin as a Hardener of Photographic Emulsions to Facilitate Staining of Epon Sections after Autoradiography

We have performed a computer assisted image analysis evaluation of the effects of two preparation protocols on morphological variables and immunolabeling density of secretory granules using rat adenohypophysis as a model. Glutaraldehyde (GA) fixation for 2 hr and chemical dehydration was compared with short GA fixation (15 min) followed by cryofixation and freeze-drying (CF-FD). The 2 hr GA fixed specimens showed spherical nuclei and secretory granules regardless of their size, contrasting with the more irregularly shaped nuclei and secretory granules after short GA-CF-FD. In the latter group of specimens a correlation could be found between smaller nuclear areas and more irregular shapes. The differences in morphological variables between the two preparation protocols might be due to a better protein stabilization and a reduced collapse of macromolecules after the 2 hr GA fixation, strengthened by the chemical dehydration. The specific immunolabeling with antiserum to growth hormone was much greater but more varied after short time GA-CF-FD than after long GA fixation. Epon surface topography over the granule area also differed: smooth after short time GA-CF-FD and furrowed after long GA fixation. Our results, demonstrating important differences in morphological parameters and immunolabeling density between two common preparation protocols, seem critical for more reliable interpretation in quantitative immunoelectron microscopy. We also emphasize the need for computer assisted image analysis and measurements in immunoelectron microscopy to ensure objective evaluations.     

Sediment Raking as a Tool for Enhancement of Phosphate and Productivity of Water in Tropical Pond System

Changes of orthophosphate in water, induced by raking of surface sediment in various ways, were compared. Twelve limnocorrals were installed in a shallow mesotrophic pond using four treatments with triplicate replication:(1) raking of surface sediment (R) by means of a rake for 5min per week (2) fish (F), by introducing six common carp and two silver carp per limnocorral, (3) raking plus fish (R+F) and (4) control (C), without raking and fishless. After day 122, treatments were reversed in limnocorrals byremoving (netting) or introducing fish and stopping or initiating raking. Samples of water and sediment were collected from each limnocorral at weekly interval and monitored for orthophosphate (OP), primary productivity, phosphate solubilizing bacteria (PSB) and sediment available phosphorus. Raking in presence of fish caused 280 and 160% increase in OP concentrations in surface and bottom water, respectively, as compared with raking in absence of fish. Net effects of fish and raking were 13-81 and 26-241%, respectively in surface water and 48-100 and 46-95% in bottom water. This implied that in bottom water, net raking effect was 13-161% greater than net fish effect. Increase in PSB population, due to raking plusfish (R+F) was 6-82% higher than increase by fish alone (F). Responses of available-P in sediment were opposite to that of water, being highest in concentration in control (C) followed by fish (F), raking (R) and raking plus fish (R+F). The primary productivity showed variations parallel with that of orthophosphate of water. This suggests that raking accelerated the transformations of available-P fromsediment to overlying water mediated through oxygen-dependent sediment water exchange mechanisms.     

Functional Morphology as a Tool in General Education

SYNOPSIS. This paper explores the proposition that in morphology,or any other scientific subject, every statement is based onmultiple assumptions already in our minds before we begin toteach or do research in the field. Progress in morphology isintimately related to its philosophical and historical foundations.Personalities, social pressures and cultural traditions alsoplay major roles in the often complex and circuitous routesof scientific discovery. General education provides a foundationfor understanding these many interactions. Morphology, itself,is a metaphor for inter-relatedness, and this metaphor can beused to explore general education in the broadest sense. Morphologyis not only a body of knowledge and processes for producingthat knowledge but also groups of people deciding on directions,collaborating on methods, and consulting on concepts. A morphologistis a member of a community of understanding in a culture composedof diverse and often conflicting influences. I suggest thatexploring these issues is an important pedagogic tool for discoveringand understanding the foundations of morphology as a scientificendeavor. This paper is appropriately dedicated to ProfessorMilton Hildebrand who understands well the interdisciplinarynature of scientific discovery.     

Fluidized Bed Hot-Melt Granulation as a Tool to Improve Curcuminoid Solubility

Curcumin is the main bioactive component of Curcuma longa L. and has recently aroused growing interest from the scientific community. Unfortunately, the medicinal properties attributed to curcuminoids are impaired by their low oral bioavailability or low solubility in aqueous solutions. Many strategies have been studied to improve curcumin solubility; however, the preparation of granules using hydrophilic materials has never been attempted. The aim of this work was to develop curcumin granules by fluidized bed hot-melt granulation using the hydrophilic carrier Gelucire® 50:13. A two-level factorial design was used to verify the influence of Gelucire® 50:13 and lactose contents found in the granules on their size, morphology, bulk and tapped densities, flow, moisture content, and water activity. The granules obtained were also evaluated by differential scanning calorimetry, thermogravimetric analysis, X-ray powder diffraction, and infrared spectrometry. The curcumin solubility and dissolution rates in water were determined by liquid chromatography. The best formulation provides an increase of curcumin solubility of 4642-fold and 3.8-fold compared to the physical mixture. The dissolution tests showed a maximum drug release from granules after 45 min of 70% at pH 1.2 and 80% at pH 5.8 and 7.4, while for non-granulated curcumin, the release was below 20% in all pH. The solid-state characterization and solubility measurement showed good stability of granules over 9 months. The results attest that the fluidized bed hot-melt granulation with hydrophilic binders is an attractive and promising alternative to obtain solid forms of curcumin with enhanced bioavailability.     

N-glycomic Profiling as a Tool to Separate Rectal Adenomas from Carcinomas

All human cells are covered by glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans. Most glycans are localized to cell surfaces and participate in events essential for cell viability and function. Glycosylation evolves during carcinogenesis, and therefore carcinoma-related glycan structures are potential cancer biomarkers. Colorectal cancer is one of the world''s three most common cancers, and its incidence is rising. Novel biomarkers are essential to identify patients for targeted and individualized therapy. We compared the N-glycan profiles of five rectal adenomas and 18 rectal carcinomas of different stages by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Paraffin-embedded tumor samples were deparaffinized, and glycans were enzymatically released and purified. We found differences in glycosylation between adenomas and carcinomas: monoantennary, sialylated, pauci-mannose, and small high-mannose N-glycan structures were more common in carcinomas than in adenomas. We also found differences between stage I–II and stage III carcinomas. Based on these findings, we selected two glycan structures: pauci-mannose and sialyl Lewis a, for immunohistochemical analysis of their tissue expression in 220 colorectal cancer patients. In colorectal cancer, poor prognosis correlated with elevated expression of sialyl Lewis a, and in advanced colorectal cancer, poor prognosis correlated with elevated expression of pauci-mannose. In conclusion, by mass spectrometry we found several carcinoma related glycans, and we demonstrate a method of transforming these results into immunohistochemistry, a readily applicable method to study biomarker expression in patient samples.Glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans, that cover all human cells. Around 1% of the human genome participates in the biosynthesis of glycans(1). This biosynthesis is the most complex post-translational modification of proteins, and the great variability in glycan structures contains a tremendous ability to fine-tune the chemical and biological properties of glycoproteins. The glycosylation process occurs most abundantly in the Golgi apparatus and the endoplasmic reticulum, but also occurs in the cytoplasm and nucleus (2). Most glycoconjugates are localized to cell surfaces, where glycans participate in events essential for cell viability and function, such as cell adhesion, motility, and intracellular signaling (2). Changes in these functions are key steps seen when normal cells transform to malignant ones, and these are also reflected in changes of a cell''s glycan profile, observed in many cancers (3, 4). Specific structural changes in glycans may serve as cancer biomarkers (5, 6), and changes in glycosylation profiles are related to aggressive behavior in tumor cells (7–9).Cancer-associated asparagine-linked glycan (N-glycan) structures may play specific roles in supporting tumor progression; growth (10, 11), invasion (12, 13), and angiogenesis (14). Changes in the N-glycan profile emerge in numerous cancers, including lung (15, 16), breast (17), and colorectal cancer (CRC)¹ (16, 18). Balog et al. (18) comparing the N-glycomic profile of CRC tissue to adjacent normal mucosa, reported differences in specific glycan structures. Moreover, serum N-glycosylation profile from patients with CRC differ from those of healthy controls (19).Colorectal cancer is the third most common cause of cancer-related death worldwide and its incidence is rising; 40% of CRCs are of rectal origin. Roughly 40% of patients have localized disease (stage I–II; Dukes A–B), another 40% loco regional disease (stage III; Dukes C), and 20% metastasized disease (stage IV; Dukes D) (20). Although stage at diagnosis is the most important factor determining prognosis, clinical outcome, and response to adjuvant treatment can markedly vary within each stage. Adjuvant therapy routinely goes to stage III patients, but the benefit of adjuvant treatment for stage II patients is unclear. Of stage II patients, 80% are cured by radical surgery alone. To identify patients who will benefit from postoperative treatment, we need novel biomarkers. The glycan profile of the tumor tissue could provide new biomarkers for diagnosis and prognosis of cancer.In this study, we characterized the N-glycomic profiles of rectal adenomas and carcinomas by MALDI-TOF mass spectrometric (MS) profiling of asparagine-linked glycans. Our aim was to identify differences between adenomas and carcinomas, and also between cancers of different stages. Based on glycan profiling, we also chose, for immunohistochemical expression studies of a series of 220 CRC patients, two glycan markers: sialyl Lewis a and pauci-mannose.     

Humanized Mouse Models as a Tool to Study Proinflammatory Cytokine Overexpression

Molecular Biology - Dysregulated proinflammatory cytokine expression may result in the development of severe pathologies, such as rheumatoid arthritis, psoriasis, and neurodegenerative diseases....     

Antibody Transfection into Neurons as a Tool to Study Disease Pathogenesis

Antibodies provide the ability to gain novel insight into various events taking place in living systems. The ability to produce highly specific antibodies to target proteins has allowed for very precise biological questions to be addressed. Importantly, antibodies have been implicated in the pathogenesis of a number of human diseases including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), paraneoplastic syndromes, multiple sclerosis (MS) and human T-lymphotropic virus type 1 (HTLV-1) associated myelopathy/tropical spastic paraparesis (HAM/TSP) ^1-9. How antibodies cause disease is an area of ongoing investigation, and data suggests that interactions between antibodies and various intracellular molecules results in inflammation, altered cellular messaging, and apoptosis ¹⁰. It has been shown that patients with MS and HAM/TSP produce autoantibodies to the intracellular RNA binding protein heterogeneous ribonuclear protein A1 (hnRNP A1) ^{3, 5-7, 9, 11}. Recent data indicate that antibodies to both intra-neuronal and surface antigens are pathogenic ^{3, 5-9, 11}. Thus, a procedure that allows for the study of intracellular antibody:protein interactions would lend great insight into disease pathogenesis.Genes are commonly transfected into primary cells and cell lines in culture, however transfection of antibodies into cells has been hindered by alteration of antibody structure or poor transfection efficiency ¹². Other methods of transfection include antibody transfection based on cationic liposomes (consisting of DOTAP/DOPE) and polyethylenimines (PEI); both of which resulted in a ten-fold decrease in antibody transfection compared to controls ¹². The method performed in our study is similar to cationic lipid-mediated methods and uses a lipid-based mechanism to form non-covalent complexes with the antibodies through electrostatic and hydrophobic interactions ¹³. We utilized Ab-DeliverIN reagent, which is a lipid formulation capable of capturing antibodies through non-covalent electrostatic and hydrophobic interactions and delivering them inside cells. Thus chemical and genetic couplings are not necessary for delivery of functional antibodies into living cells. This method has enabled us to perform various antibody tracing and protein localization experiments, as well as the analyses of the molecular consequences of intracellular antibody:protein interactions ⁹.In this protocol, we will show how to transfect antibodies into neurons rapidly, reproducibly and with a high degree of transfection efficiency. As an example, we will use anti-hnRNP A1 and anti-IgG antibodies. For easy quantification of transfection efficiency we used anti-hnRNP A1 antibodies labelled with Atto-550-NHS and FITC-labeled IgG. Atto550 NHS is a new label with high molecular absorbtion and quantum yield. Excitation source and fluorescent filters for Atto550 are similar to Cy3 (Ex. 556 Em. 578). In addition, Atto550 has high photostability. FITC-labeled IgG were used as a control to show that this method is versatile and not dye dependent. This approach and the data that is generated will assist in understanding of the role that antibodies to intracellular target antigens might play in the pathogenesis of human diseases.