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PubMed2006

Distinctions between separation anxiety and social anxiety in children and adolescents.

Separation anxiety and social phobia are intertwined to a considerable degree, and high comorbidity rates have been reported. The present study used latent class analysis (LCA) to investigate if classes of children and adolescents with-simultaneously-high rates of separation anxiety and low rates of social anxiety symptoms, or vice versa, could be identified. Eight- to 18-year-olds from a large general population (n=1000) and referred sample (n=735) were assessed with the Multidimensional Anxiety Scale for Children (MASC). With LCA, a separate class of referred 8-11-year-old children with high separation anxiety scores, and simultaneously lower social anxiety scores was identified, next to a class of children with high scores on separation anxiety and social anxiety. In the other groups (referred 12-18-year-olds and children and adolescents from the general population), a class with individuals who specifically scored high on separation anxiety could not be revealed. The results indicated that separation anxiety represents a different construct than social anxiety in referred children (but not in referred adolescents or in the general population). It can be concluded that, in referred children, research regarding etiology and treatment outcome of anxiety symptoms should be aimed specifically at separation anxiety and social anxiety, instead of just investigating a broader anxiety dimension.

Adolescent↗
PubMed2005

Separation of sperm and epithelial cells in a microfabricated device: potential application to forensic analysis of sexual assault evidence.

Forensic DNA analysis of sexual assault evidence requires separation of DNA from epithelial (victim) and sperm (perpetrator) cells. The conventional method used by crime laboratories, which is termed "differential extraction", is a time-consuming process. To supplant the conventional process, separation of sperm from a biological mixture containing epithelial cells has been demonstrated on a microfluidic device. This separation utilizes the differential physical properties of the cells that result in settling of the epithelial cells to the bottom of the inlet reservoir and subsequent adherence to the glass substrate. As a result, low flow rates can be used to separate the sperm cells from the epithelial cell-containing biological mixture. Following cell separation on the microdevice, DNA extraction, amplification, and separation were performed using conventional laboratory methods, showing that the cell separation product in the outlet reservoir was of male origin. The reported cell separation has the potential to impact the forensic DNA analysis backlog of sexual assault cases by circumventing the time-consuming conventional differential extraction procedure.

Cell Separation↗
PubMed2000

Cell separation by dielectrophoretic field-flow-fractionation.

Dielectrophoretic field-flow-fractionation (DEP-FFF) was applied to several clinically relevant cell separation problems, including the purging of human breast cancer cells from normal T-lymphocytes and from CD34+ hematopoietic stem cells, the separation of the major leukocyte subpopulations, and the enrichment of leukocytes from blood. Cell separations were achieved in a thin chamber equipped with a microfabricated, interdigitated electrode array on its bottom wall that was energized with AC electric signals. Cells were levitated by the balance between DEP and sedimentation forces to different equilibrium heights and were transported at differing velocities and thereby separated when a velocity profile was established in the chamber. This bulk-separation technique adds cell intrinsic dielectric properties to the catalog of physical characteristics that can be applied to cell discrimination. The separation process and performance can be controlled through electronic means. Cell labeling is unnecessary, and separated cells may be cultured and further analyzed. It can be scaled up for routine laboratory cell separation or implemented on a miniaturized scale.

Animals↗
PubMed2026

High-throughput identification of endogenous biomolecular condensates and phase-separating proteins.

Biomolecular condensates formed through liquid-liquid phase separation regulate cellular processes, and their dysregulation causes disease. Current methods for identifying endogenous phase-separating proteins have low throughput and cannot capture dynamic responses to stimuli. Here we present a protocol combining osmotic compression or transforming growth factor-β (TGF-β) treatment to induce condensation with sucrose density gradient centrifugation and quantitative mass spectrometry to enable systematic, high-throughput identification of endogenous condensates and phase-separating proteins. The method exploits the density changes that occur when phase-separating proteins undergo oligomerization during condensate formation. In H1975 cells, we identified over 1,500 phase-separating proteins under osmotic compression or TGF-β treatment; 538 of these candidates were not present in PhaSepDB, a database that compiles in vivo, in vitro and omics-derived proteins. The approach detects constitutive condensates and proteins that dynamically phase-separate in response to osmotic stress or TGF-β signaling. This protocol provides proteome-wide analysis of fractions of proteins having different densities and enables temporal resolution of phase-separation events. The procedure takes ~9 d and requires expertise in cell culture, biochemistry and mass spectrometry. This method enables systematic study of biomolecular condensates and disease-associated phase-separation mechanisms.

Phase Separation↗
PubMed1993

Positive selection of human blood cells using improved high gradient magnetic separation filters.

High gradient magnetic separators (HGMS) create magnetic field gradients that can be used to attract much smaller and less magnetic particles than those required for conventional magnetic separation techniques. As a result cells can be labeled with submicron magnetic particles and still be separated using an HGMS filter. Typically, HGMS filters consist of random arrays of wire such as stainless steel wool. Wire elements arranged regularly in a filter should allow more efficient separation of cells. Filters were constructed containing ordered wire arrays composed of 430 series stainless steel wire mesh with wire diameters of 50, 100, or 150 microns. The ability of these filters to separate T cells from peripheral blood mononuclear cell suspensions was tested and found superior to random arrays of 302 series stainless steel wire (Thomas et al, 1992). Target cells recognized by OKT5 monoclonal antibody were cross-linked to dextran-iron particles of approximately 20 nm in diameter. Separation conditions were optimized and after one passage through the filter 88% of the OKT5+ cells were recovered in the enriched fraction with 85% purity (%OKT5+). Multiple passages (3 times) could achieve 99% purity with 68% recovery. Variations in separation flow rate had a large effect on the balance between purity and recovery. Optimum separation efficiencies were achieved only when > 10(8) cells were processed. The primarily cause of nonspecific entrapment of CD8- cells was not nonspecific magnetic labeling of cells but the physical (nonmagnetic) characteristics of the filter/filter chamber.

Adult↗
PubMed1991

Separation of leucocytes: improved cell purity by fine adjustments of gradient medium density and osmolality.

This paper briefly reviews commonly used procedures for separation of mononuclear cells (MNC) and granulocytes from human blood with X-ray contrast media as gradient material, and also presents new and modified procedures for leucocyte preparation. Standard techniques for human blood do not always yield satisfactory results with blood from other species. In general pure MNC are easily obtained (top fraction), but often the granulocyte fraction has a low purity, due to contamination with MNC that move to the bottom during centrifugation and contaminate the granulocyte suspension. Obviously the density distribution of MNC differs between species. However, the separation can be improved by fine adjustment of gradient medium osmolality. For this purpose we have used Nycodenz, a non-ionic X-ray contrast medium. A favourable property of Nycodenz solutions is that the osmolality and density can easily be varied over a broad range. The cells react promptly to a change of medium osmolality. In hypertonic medium the cells expel water, shrink, their density increases and they sediment faster, in spite of a smaller radius. Further, the cells may pass what was initially a density barrier. A hypotonic environment has the opposite effect. In the present work we were able to show that a slight change of medium osmolality clearly improved different techniques for separation of leucocyte subgroups. For instance, the Isopaque-Ficoll (IF) technique consistently yielded MNC and granulocytes of high purity with human blood. However, with blood from rabbits, rats and mice the granulocyte suspensions were contaminated by 40-60% MNC. By utilizing Nycodenz, and lowering the osmolality by 10-12 per cent (at constant density--1.077 g/ml) we obtained satisfactory separation of MNC as well as granulocytes with blood from these species. A problem in the routine separation of granulocytes (IF) is a high contamination of erythrocytes (2-5 per cell) in the granulocyte suspension. With a two-layer technique with Nycodenz solutions of different densities it was possible to separate granulocytes almost devoid of erythrocytes, after proper adjustment of osmolality. By appropriate combination of density and osmolality, Nycodenz was a suitable gradient material in other separation procedures as well, e.g. the separation of monocytes and mast cells. To facilitate the use of Nycodenz as a versatile gradient material, a computer program providing recipes for various Nycodenz solutions is included as an appendix.

Animals↗
PubMed2006

Prevalence and correlates of estimated DSM-IV child and adult separation anxiety disorder in the National Comorbidity Survey Replication.

OBJECTIVE: Despite its inclusion in DSM-IV, little is known about the prevalence or correlates of adult separation anxiety disorder or its relationship to the childhood disorder. Results of the first epidemiological study of adult separation anxiety disorder, to the authors' knowledge, and its relationship to childhood separation anxiety disorder are presented. METHOD: Data were from the National Comorbidity Survey Replication (NCS-R), a nationally representative survey of U.S. households. A fully structured, lay-administered diagnostic interview assessed a wide range of DSM-IV disorders, including separation anxiety disorder. No independent clinical validation was obtained of the assessment. RESULTS: Lifetime prevalence estimates of childhood and adult separation anxiety disorders were 4.1% and 6.6%, respectively. Approximately one-third of the respondents who were classified as childhood cases (36.1%) had an illness that persisted into adulthood, although the majority classified as adult cases (77.5%) had first onset in adulthood. The assessment of separation anxiety disorder in the NCS-R was comorbid with other NCS-R or DSM-IV disorders and associated with severe role impairment in roughly half of the comorbid cases and one-fourth of the pure cases. The majority of people with estimated adult separation anxiety disorder are untreated, even though many obtain treatment for comorbid conditions. CONCLUSIONS: Criteria for adult separation anxiety disorder should be refined in future editions of DSM because the disorder is likely to be much more common in adults than previously recognized. Research is needed to develop and evaluate treatments that take into consideration its high comorbidity with other DSM-IV disorders.

Adolescent↗
PubMed2004

What effect does classroom separation have on twins' behavior, progress at school, and reading abilities?

We investigated the effects of classroom separation on twins' behavior, progress at school, and reading abilities. This investigation was part of a longitudinal study of a nationally-representative sample of twins (the E-risk Study) who were assessed at the start of school (age 5) and followed up (age 7). We examined three groups of twins: pairs who were in the same class at both ages; pairs who were in separate classes at both ages; and pairs who were in the same class at age 5, but separated by age 7. When compared to those not separated, those separated early had significantly more teacher-rated internalizing problems and those separated later showed more internalizing problems and lower reading scores. Monozygotic (MZ) twins showed more problems as a result of separation than dizygotic (DZ) twins. No group differences emerged for externalizing problems, ADHD or prosocial behaviors. The implications of the findings for parents and teachers of twins, and for school practices about separating twins, are discussed.

Anxiety, Separation↗
PubMed1987

[The effects of mother-infant separation upon nursing and suckling behaviors of the rat].

The present study was intended to evaluate the hypothesis that the nursing-suckling relationship of rats proceeds from the mother-leading-type in early postpartum days to the infants-leading-type in later postpartum. Four groups were organized to make tests on the effects of different kinds of mother-infant separation (8-hr) upon nursing-suckling behaviors. Groups were Groups MIS (a separated mother and infants group), MS (mother-separated and infants-fostered group), IS (a foster-mother and separated-infants group), and C (non-separated control group). The separation of each group, lasting for 8-hr, was made at 1-2, 6-7, 11-12, 16-17, 21-22, 26-27, and 31-32 days after parturition. In Exp. I, nursing behavior of mother and suckling and/or eating behavior of infants in mother-infant interactions were observed by time-sampling method for 30 min following 8-hr of separation. In Exp. II, the suckling test of infants (half of the litter) was made on the anesthetized mother at the presence of food pellets. The results of Exp. I showed that nursing-suckling episodes were more frequent in Groups MIS, MS, and IS than in Group C (fig. 1). Further, the differences among the first three groups were not significant before 11-12 days of age. However, the infant-separated group (Group IS) showed higher level of response at 16-17, 21-22 days of age, suggesting the active role of infants in starting nursing-suckling episodes at these ages.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed2003

The impact of father absence on adolescent separation-individuation.

The authors compared separation-individuation and psychological separation from fathers of 25 adolescent boys who were living with both biological parents with that of 25 boys who were living with their biological mothers in homes in which the fathers did not reside. The results showed that the boys in the 2 groups did not differ on measures of separation-individuation and that the quality of the mother-son relationship mediated several of the assessed separation-individuation manifestations. The authors' initial data analysis with regard to psychological separation showed that boys who lived in homes in which the father was a nonresident were more separated on 2 of the 4 dimensions assessed; however, when controlled for quality of mother-son and father-son relationship, these differences were not significant. The frequency of father contact in homes in which the father did not reside was positively correlated with healthy separation but negatively correlated with functional, attitudinal, and emotional independence from the father. The results of the study support the importance of the quality of a child's relationship with his or her mother and father as a mediator of several dimensions of the separation-individuation process.

Adolescent↗
PubMed1990

Density-gradient separation for 4-hydroperoxycyclophosphamide purging of autologous bone marrow grafts.

We studied density-gradient separation of autologous bone marrow grafts in preparation for ex vivo 4-hydroperoxy-cyclophosphamide (4-HC) purging. A two-step procedure of buffy coat isolation followed by Ficoll-diatrizoate separation was used. Buffy-coat cells were isolated primarily either using the COBE 2991 Blood Cell Processor or Haemonetics 30 Cell Separator. All density-gradient separations were performed using the COBE 2991. The nucleated cell recoveries after buffy-coat isolation were 44.3 +/- 10.4% and 85.1 +/- 10.5% (+/- standard deviation, P less than 0.001) for the Haemonetics 30 and COBE 2991 isolated grafts, respectively. The final nucleated cell recoveries after density-gradient separation for these two buffy-coat techniques were 24.2 +/- 10.1% and 29.5 +/- 14.7% (p = 0.30). Compared to manual density-gradient separation, the techniques using the COBE 2991 recovered slightly higher numbers of nucleated cells and CFU-GM. The red blood cell concentration of the 4-HC incubation mixture was 1.0 +/- 0.6% and the CFU-GM survival after treatment was 2.4 +/- 2.6%. The variability in CFU-GM survival after 4-HC incubation of the density-gradient separated grafts appears less than previous experience treating buffy-coat cells. Density-gradient separation of autologous bone marrow appears to provide a more uniform treatment with 4-HC and may improve the clinical transplant course of the autograft recipient.

Bone Marrow↗
PubMed1988

[Separation and functional analysis of tumor-infiltrating lymphocytes in brain tumor tissues].

Separation of tumor-infiltrating lymphocytes (TIL) from surgical specimen of fourteen gliomas and 6 metastatic brain tumor tissues was achieved by discontinuous density gradients. Comparison of phenotypes between separated lymphocytes and TIL in situ was examined by immunohistochemical method using monoclonal antibodies. The cytotoxic activity of these separated lymphocytes was also examined in short-term 51Cr release assay against fresh autologous tumor cells. Immunohistochemical staining of separated lymphocytes revealed the preservation of cell surface antigens of the lymphocytes. Most of separated lymphocytes were T lymphocytes and both phenotypes cytotoxic/suppressor and helper/inducer T lymphocytes were found. The Leu-3a/2a ratio of separated lymphocytes were compared with TIL in situ in the same patients and these values were almost equal in both lymphocytes. Cell number of separated lymphocytes were 1.2 x 10(5)-2.1 x 10(5)/g in glioma and 1.1 x 10(5)-3.8 x 10(5)/g in metastatic patients. Cytotoxicity against autologous tumor cells was detected in 5 out of 12 glioma and 2 out of 6 metastatic patients. There was no correlation between the cytotoxicity and cell number of separated lymphocytes or HLA-DR antigen expression on tumor cells.

Adolescent↗
PubMedDate not supplied

Influence of neutrophil separation on the expression of adhesion molecules.

In many assays of polymorphonuclear neutrophil (PMN) function the first step is separation of PMN from whole blood. In the present investigation it was examined if PMN separation leads to an altered expression of neutrophil surface membrane adhesion molecules. Samples have been taken from 20 healthy volunteers (10 male, 10 female; 39.7 +/- 11.8 years of age). PMN activation was measured cytometrically using the following antibodies against PMN surface membrane receptors: L-selectin (CD 62 L), beta-2-integrin Mac-1 (CD 11b) and Intercellular Adhesion Molecule 1 (CD54). PMN activation was determined in whole blood and after separation of PMN using density gradients. After PMN separation all three adhesion molecules appeared increased but the effect was only statistically significant for CD 54 (Wilcoxon test). Data (mean fluorescence intensity in arbitrary units) were: CD 62 L: 62 +/- 37 in whole blood, 82 +/- 28 after separation; p = 0.0674, CD 11b: 94 +/- 55 in whole blood, 111 +/- 47 after separation; p = 0.1454, CD 54; 13 +/- 12 in whole blood, 81 +/- 35 after separation; p < 0.0001. With the present data available it can be assumed that separation of PMN from whole blood can influence the results of flow cytometric assays.

Adult↗
PubMed1999

Separation and characterization of barley (Hordeum vulgare L.) hordeins by free zone capillary electrophoresis.

Extraction conditions, separation conditions, and capillary rinsing protocols were optimized for the separation of barley hordeins by free zone capillary electrophoresis. Stable hordein extracts were obtained with a single 5 min extraction after the albumins and globulins were removed. Hordeins had to be reduced for optimal resolution. Optimum separation conditions for hordein separations were 100 mM phosphate-glycine buffer containing 20% acetonitrile and 0.05% hydroxypropylmethylcellulose. The addition of zwitterionic sulfobetaine detergents containing hydrocarbon tails of eight and ten carbons slightly improved the resolution of the separations, but not enough to warrant their use on a routine basis. The migration positions of the hordein subclasses were determined by two- dimensional reversed-phase high-performance liquid chromatography x free zone capillary electrophoresis mapping. The hordein subclasses formed clusters similar to those of wheat gliadins. Separation-to-separation repeatability was good, with migration time relative standard deviations < 1% for a 15-run period. For routine discrimination of cultivars, a 2 min post-separation rinse with 500 mM acetic acid was necessary to prevent protein build-up on the capillary walls. An example of successfully differentiating barley cultivars using this technique is shown.

Chromatography, High Pressure Liquid↗
PubMed2000

Separation of different physical forms of plasmid DNA using a combination of low electric field strength and flow in porous media: effect of different field gradients and porosity of the media.

The retention of different physical forms of DNA by an electric field in a chromatography system was studied. We were able to effectively separate the supercoiled and the open circular forms of plasmid DNA using this type of electrochromatography system. Chromatography columns were packed with porous beads, and an axial electric field was applied so that convective buffer flow opposed the direction of electrophoresis of the DNA. A model system composed of approximately equal amounts of the super-coiled and open circular forms of the plasmid pBR 322 (4322 base pairs) was used to test the separation. Chromatography beads (agarose-based) with different porosities were used to determine the effect of the stationary phase on the separation. The porous media did not have a major effect on the separation, but the best separations were obtained using porous chromatography media made with the highest agarose concentration (10% agarose). Selective elution of plasmid DNA with different forms was obtained by either increasing the flow rates or decreasing the electric field strength (by steps or a gradient). In all the separations, the more compact supercoiled form of the plasmid was retained less strongly than either the open circular form (nicked) or the linear form. High molecular weight host genomic DNA was more strongly retained than the plasmid DNA. Increasing the ionic strength of the buffer improved resolution and capacity. The capacity of the separation was determined by injecting increasing amounts of plasmid DNA. Satisfactory separation was obtained at sample loading of up to 360 microg of total DNA on a column with dimensions of 2.5 by 11 cm (bed volume of 54 mL). The retention of DNA depends upon a counter-current flow of electrophoresis and convective flow and could be regarded as a type of field flow fractionation. The retention of the DNA by the electric field and flow is discussed in relation to the diffusion coefficients of the DNA.

Buffers↗
PubMed2000

Electrofilament deposition and off-column detection of analytes separated by capillary electrophoresis.

Capillary electrophoresis interfaced with electrospray is a convenient technique for continuously transferring column effluent from capillary-to-planar format. Conditions are optimized to produce a narrow (approximately 20 microm) liquid filament (electrofilament), which is capable of depositing spatially focused bands with track widths that are routinely 100 microm. A fiber optic-based, laser-induced fluorescence cell is employed to monitor the separation on-column while the separated bands are deposited onto a moving substrate. The photodetection of deposited bands is accomplished by using either a charge-coupled device camera or a photomultiplier tube. Deterioration of on-column separation performance is observed when the electrofilament voltage is applied. Elevating the inlet of the capillary column, to provide hydrodynamic flow, restores separation performance. Substrate temperature and translational rates are optimized with respect to both off-column separation efficiency and signal intensity. Off-column separation efficiencies of 65 000 plates per meter were achieved. A linear dynamic range of 10(3) and a limit of detection of 10(-8) M were obtained for kiton red deposited onto a reversed phase thin-layer chromatography plate. To demonstrate the applicability of this technique to more complex separation solutions, a dye mixture was successfully separated and deposited with sodium dodecyl sulfate in the running buffer.

Electrophoresis, Capillary↗
PubMed2000

Separation of a group of N-phenylpyrazole derivatives by micellar electrokinetic chromatography: application to the determination of solute-micelle association constants and estimation of the hydrophobicity.

Micellar electrokinetic chromatography (MEKC) was applied to the separation of a group of N-phenylpyrazole derivatives. Sodium dodecyl sulfate (SDS) as micellar system and 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) as separation buffer (pH 10) were employed in the absence and presence of different percentages of medium chain alcohols (n-propanol or n-butanol). The separation of multicomponent mixtures of the solutes studied enabled the rapid determination of their retention factors which, in turn, allowed the study of the separation selectivity of compounds and the determination of their solute-micelle association constants (from the linear variation of the retention factors as a function of the total surfactant concentration in the separation buffer). Separation selectivity was studied according to the elution range and number of solutes separated in all the electrolyte solutions employed (45 micellar phases). The effect of the buffer concentration (0.05, 0.08 and 0.10 M), the alcohol nature (n-propanol or n-butanol) and the alcohol percentage (1, 3 or 5%) of the values obtained for the solute-micelle association constants was also studied. The best separation (12 solutes) was performed when a 0.08 M CHES buffer, pH 10, 0.02 M SDS modified by 5% n-butanol was used. The possibilities of using MEKC for evaluating the hydrophobicity of compounds was investigated through the study of the correlation between the logarithm of the retention factors of N-phenylpyrazole derivatives and their logarithm of the octanol-water distribution coefficients estimated by high performance liquid chromatography (HPLC).

Alcohols↗
PubMed2006

Strategy for selection of methods for separation of bioparticles from particle mixtures.

The desired product of bioprocesses is often produced in particulate form, either as an inclusion body (IB) or as a crystal. Particle harvesting is then a crucial and attractive form of product recovery. Because the liquid phase often contains other bioparticles, such as cell debris, whole cells, particulate biocatalysts or particulate by-products, the recovery of product particles is a complex process. In most cases, the particulate product is purified using selective solubilization or extraction. However, if selective particle recovery is possible, the already high purity of the particles makes this downstream process more favorable. This work gives an overview of typical bioparticle mixtures that are encountered in industrial biotechnology and the various driving forces that may be used for particle-particle separation, such as the centrifugal force, the magnetic force, the electric force, and forces related to interfaces. By coupling these driving forces to the resisting forces, the limitations of using these driving forces with respect to particle size are calculated. It shows that centrifugation is not a general solution for particle-particle separation in biotechnology because the particle sizes of product and contaminating particles are often very small, thus, causing their settling velocities to be too low for efficient separation by centrifugation. Examples of such separation problems are the recovery of IBs or virus-like particles (VLPs) from (microbial) cell debris. In these cases, separation processes that use electrical forces or fluid-fluid interfaces show to have a large potential for particle-particle separation. These methods are not yet commonly applied for large-scale particle-particle separation in biotechnology and more research is required on the separation techniques and on particle characterization to facilitate successful application of these methods in industry.

Biological Products↗