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PubMed2003

Chiral separation of 2,3-allenoic acid by capillary zone electrophoresis using cyclodextrin derivatives.

In this paper, five of six samples of 2,3-allenoic acid enantiomers were separated by capillary zone electrophoresis (CZE) using hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) as chiral selectors. Using HP-beta-CD for chiral separation, three of the six enantiomers were separated. Five experimental conditions including HP-beta-CD concentration, pH, buffer concentration, temperature, and running voltage were investigated for their influence on separation and migration using enantiomers of 2-methyl-4-phenyl-2,3-butadienoic acid (A) and 2-(n-propyl)-4-phenyl-2,3-butadienoic acid (B) as samples. Good separation results were observed when [HP-beta-CD] = 3-12 mmol/L and pH = 7-9 for samples A and B. The temperature range of 15-25 degrees C can be selected for convenience. According to the chiral separation results, HP-beta-CD and HP-gamma-CD should be valuable selectors to separate 2,3-allenoic acids and HP-gamma-CD was suggested to separate the 2,3-allenoic acid samples with a group at 4-position bulkier than phenyl.

Journal Article↗
PubMed1991

Time-dependent effects of peer separation on lymphocyte proliferation responses in juvenile squirrel monkeys.

This study evaluated the immunological, hormonal, and behavioral responses of juvenile squirrel monkeys to repeated social separations of varying length. Following a 3-hr separation, lymphocyte responses to stimulation with the mitogen concanavalin A (Con A) declined significantly, and these alterations were sustained after a 24-hr separation period. The responses to Con A and to a second mitogen, phytohemagglutinin (PHA), were suppressed after 2 days. At the end of a 7-day separation period, immune responses were not significantly different from basal values. Plasma cortisol levels were elevated above basal levels in all animals after the 3-hr, 24-hr, and 2-day separations, but were not elevated after the 7-day separation. While we observed no statistically significant changes in locomotor activity or calling behavior during any of the separations, the monkeys tended to be most active and to call most immediately following separation. Our findings concur with earlier reports indicating that social stressors can influence lymphocyte proliferation in nonhuman primates and that certain cell types might be differentially sensitive to stress, but also indicate that these influences are transient.

Animals↗
PubMed1995

Total cortisol, free cortisol, and growth hormone associated with brief social separation experiences in young macaques.

Many behavioral, immunological, and physiological consequences or brief maternal separation in bonnet (Macaca radiata) and pigtail monkeys (Macaca nemistrina) have been documented. However, the impact of social separation on plasma cortisol and growth hormone is unknown for these particular species. In the present study, the behavioral and endocrinological consequences of a 2-week maternal separation in socially housed infant bonnet and pigtail monkeys were followed. In seven pairs (separated and matched control) of bonnet and six pairs of pigtail infants, plasma was obtained under baseline, separated, and reunion conditions twice weekly for the duration of the study. Blood samples were obtained from both infants of the pair in approximately 10 min. Plasma total cortisol, free cortisol, and growth hormone were measured in these samples. Focal animal behavioral observations were made on all subjects twice daily throughout the study period. In both species, total cortisol and free cortisol rose immediately following maternal separation in comparison to the matched nonseparated controls and returned to basal levels (e.g., that of matched nonseparated controls) following reunion with the mother. In contrast, plasma growth hormone rose only in the pigtail infants over a time course that peaked around the time of reunion. Multiple regression techniques indicated for the first week of separation, in the separated but not control subjects, that mean plasma free and total cortisol was positively related to distress behaviors (vocalization and postural slouch) observed during this week and negatively related to social behaviors (play and proximity to others) noted during the same period. In contrast, plasma growth hormone was related to both species and sex of the subjects but unrelated to behavioral variables.

Animals↗
PubMed1997

Versatile low-viscosity sieving matrices for nondenaturing DNA separations using capillary array electrophoresis.

The high-resolution separation of double-stranded DNA (dsDNA) has important applications in physical mapping strategies and in the analysis of polymerase chain reaction (PCR) products. Although high-resolution separations of dsDNA by capillary electrophoresis (CE) have been reported, pulsed fields were required to achieve complete resolution of DNA fragments beyond 23 kilobase pairs (kbp). Here, we report a single formulation to separate a broad range (80 bp-40 kbp) of DNA fragments without the use of pulsed fields. We used a low-viscosity sieving medium (ca. 5 cP, at 25 degrees C) based on polyethyleneoxide (PEO) to separate DNA fragments up to 40 kbp. The matrix contained a mixture of 0.5% PEO (Mn 10(6)) to separate fragments up to 1.5 kbp, combined with 0.1% PEO (Mn 8 x 10(6)) to separate fragments between 1-40 kbp, within a single run. All PEO matrix formulations tested were compatible with a variety of intercalating dyes and with two different capillary wall coating methods. We obtained a detection limit of 25 fg of a 200 bp DNA quantitation standard using Vistra Green in the matrix. Resolution was best using short injection times (5 s or less) and low field strengths (approximately 100 V/cm). Sample runs were complete in 70 min, and use of the capillary array electrophoresis (CAE) system permitted high-throughput DNA analysis. The size range separated is approximately 10 times greater than with conventional slab gel separations.

Coloring Agents↗
PubMed1998

Exploitation of a monoclonal antibody for weak affinity-based separation in capillary gel electrophoresis.

Weak biospecific recognition has been established for affinity separation in high performance liquid chromatography (HPLC). The use of weak affinity chromatography (WAC) has been limited previously by the insufficient separation efficiency achieved, allowing only some 1000 plates/m to be obtained. However, it has been shown that chiral drug separation can be performed with capillary affinity gel electrophoresis (CAGE) at considerably improved efficiency as compared with traditional chromatographic procedures. Our present study demonstrates the potential of weak affinity monoclonal antibodies as a generic method for immunologically based separations in capillary electrophoresis. Monoclonal antibodies were polymerized within a silica capillary and were used for the separation of structurally similar carbohydrate antigens. The results indicate that weak biospecific interactions can be utilized in a CAGE format to produce highly selective separation of the alpha- and beta-forms of p-nitrophenyl-labeled maltose. It remains to be seen, however, how efficient weak affinity separation in CAGE can be compared with affinity HPLC protocols. Details of typical separations and of the preparation of the antibody gel are presented.

Antibodies, Monoclonal↗
PubMed1998

Mixed-mode separation of polycyclic aromatic hydrocarbons (PAHs) in electrokinetic chromatography.

A mixed-mode separation technique has been developed and optimized for the separation of the 16 Environmental Protection Agency (EPA) priority polycyclic aromatic hydrocarbons (PAHs). The procedure utilized two different buffer additives as pseudo-stationary phases with different selectivities towards the analytes. Sodium dioctyl sulfosuccinate (DOSS) displayed selectivities for PAHs which were somewhat similar to the C18 phase in reversed-phase high performance liquid chromatography (HPLC). High acetonitrile content required for an effective separation prevented the formation of micelles as confirmed by fluorescence spectroscopy. Consequently, the separation could be attributed to the solvophobic association of the PAH molecules with hydrophobic chains of the DOSS surfactant. In another mode of separation, sulfobutylether-beta-cyclodextrin (SB-beta-CD) separated the 16 PAHs on the formation of inclusion complexes with the PAHs, and exhibited different selectivities for the PAHs compared to DOSS. SB-beta-CD and DOSS were then combined in the running buffer to form a mixed pseudo-stationary phase for the separation of the 16 PAHs. Due to the different selectivities of SB-beta-CD and DOSS for the PAHs, the separation of the 16 PAHs was appreciably improved compared to that using DOSS or SB-beta-CD alone. All the 16 PAHs were baseline-resolved using an optimized running buffer containing 22.5 mM DOSS, 15 mM SB-beta-CD, 15% acetonitrile and 5 mM hydroxypropyl-beta-cyclodextrin in 6 mM borate at pH 9.

Chromatography↗
PubMed2004

Dynamics of interconversion of enantiomers in chiral separation systems: a novel approach for determination of all rate constants involved in the interconversion.

When enantiomers separated by chromatography or capillary electrophoresis undergo interconversion reaction (enantiomerization) during the separation, it leads to a typical detection pattern: two individual peaks of the separated enantiomers are connected with a plateau consisting of a mixture of both separated enantiomers. We propose a separation method for determination of all individual rate constants (or inversion barriers) of the interconversion. The method enables to distinguish which part of interconversion takes place in the free (unbound) form of the analyte and which part in the complexed (bound) form. Further, we propose a complete dynamic model of capillary electrophoresis of interconverting enantiomers based on solving a complete set of continuity equations for all constituents of the separation system together with complexation and acid-base equilibria. This allows a simulation of both linear and nonlinear mode of separation and understanding all processes taking place in such enantioseparation systems. We demonstrate the applicability of the method on determination of the rate constants of interconversion of oxazepam enantiomers separated in systems with charged cyclodextrin chiral selectors.

Acid-Base Equilibrium↗
PubMed2004

Phospholipid-lysozyme coating for chiral separation in capillary electrophoresis.

A phospholipid coating with lysozyme as chiral recognition reagent permeated into the phospholipid membrane was developed for the chiral capillary electrophoretic (CE) separation of D- and L-tryptophan. As a kind of carriers, coated as phospholipid membranes onto the inner wall of a fused-silica capillary, liposomes are able to interact with basic proteins such as lysozyme, which may reside on the surface of the phospholipid membrane or permeate into the middle of the membrane. The interaction results in strong immobilization of lysozyme in the capillary. Coatings prepared with liposomes alone did not allow stable immobilization of lysozyme into the phospholipid membranes, as seen from the poor repeatability of the chiral separation. When 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide (M1C4) was applied as a first coating layer in the capillary, the electroosmotic flow (EOF) was effectively suppressed, the phospholipid coating was stabilized, and the lysozyme immobilization was much improved. The liposome composition, the running buffer, and the capillary inner diameter all affected the chiral separation of D- and L-tryptophan. Coating with 4 mM M1C4 and then 1 mM phosphatidylcholine (PC)/phosphatidylserine (PS) (80:20 mol%), with 20 mM (ionic strength) Tris at pH 7.4 as the running buffer, resulted in optimal chiral separation with good separation efficiency and resolution. Since lysozyme was strongly permeated into the membrane of the phospholipids on the capillary surface, the chiral separation of D- and L-tryptophan was achieved without lysozyme in the running buffer. The effects of different coating procedures and separation conditions on separation were evaluated, and the M1C4-liposome and liposome-lysozyme interactions were elucidated. The usefulness of protein immobilized into phospholipid membranes as a chiral selector in CE is demonstrated for the first time.

Electrophoresis, Capillary↗
PubMed2005

Fast separation of single-stranded oligonucleotides by capillary electrophoresis using OliGreen as fluorescence inducing agent.

The fast separation of oligonucleotide (oligos) sizing marker by CE using OliGreen and including effects due to the concentration of separation medium and urea denaturant is presented. OliGreen dye is found to be more sensitive than ethidium bromide (by a factor of about 6 based on S/N considerations) for the oligos' separations. Higher concentration of F127 in 1xTris-boricacid-EDTA (TBE) up to 30% w/v leads to better resolution of oligos separations. The addition of urea into the separation medium decreases the sensitivity. With an optimized running condition, the oligos sizing marker could be successfully separated with 1-base resolution within 1.3 min by using 30% w/v F127/1xTBE solution as the separation medium at an applied electric field of 800 V/cm in a 3 cm long capillary, the fastest capillary gel electrophoresis separation with high resolution reported to date for oligos in the similar size range.

Buffers↗
PubMed2005

Investigation of solvent effects in capillary electrophoresis for the separation of biological porphyrin methyl esters.

The effects of organic solvents on the capillary electrophoresis (CE) separation of a number of important biological porphyrin methyl esters - six weakly basic, hydrophobic cyclic tetrapyrroles possessing two and four to eight methyl ester groups around the periphery of the porphyrin ring - were investigated in the mode of micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic chromatography (MEEKC), and nonaqueous CE. In aqueous MEKC, partial separation of the six neutral porphyrin methyl esters was obtained with an organic modifier (acetonitrile) in the concentration range between 20 and 40%, in which sodium dodecyl sulfate (SDS) molecules might be present in the form of SDS micelles and/or SDS micelle-like aggregates. Relatively stable SDS micelles can be formed in nonaqueous MEKC using formamide as the separation medium, but the separation of the target analytes remained unsatisfactory. Improved resolution of all six porphyrin methyl esters was obtained using MEEKC with the running buffer consisting of 0.8% w/w n-heptane (oil phase), 2.25% w/w SDS and 1.0% w/w Brij 35 (mixed surfactant), 6.6% w/w 1-butanol (cosurfactant), and 30% v/v 2-propanol (second cosurfactant), but reproducibility in terms of peak areas for certain porphyrins (especially uroporphyrin I octamethyl ester) was found to be very poor. Best separation performances were achieved with nonaqueous CE separations in which the weakly basic porphyrin methyl esters were protonated under strongly acidic conditions (e.g., using 10 mM perchloric acid) in mixed organic solvents. For example, using a 50:50 mixture of methanol and acetonitrile as the separation medium, baseline separation of all six (positively charged) porphyrin methyl esters can be obtained within 3 min and the average precision (RSD, N = 13) in terms of migration time and peak area were 0.55 and 2.16%, respectively.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2002

Adhesive phase separation at the dentin interface under wet bonding conditions.

Under in vivo conditions, there is little control over the amount of water left on the tooth and, thus, there is the danger of leaving the dentin surface so wet that the bonding resin undergoes physical separation into hydrophobic and hydrophilic-rich phases. The purpose of this study was to investigate phase separation in 2,2-bis[4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl] propane (BisGMA)-based adhesive using molecular microanalysis and to examine the effect of phase separation on the structural characteristics of the hybrid layer. Model BisGMA/HEMA (hydroxyethl methacrylate) mixtures with/without ethanol and commercial BisGMA-based adhesive (Single Bond) were combined with water at concentrations from 0 to 50 vol%. Macrophase separation in the BisGMA/HEMA/water mixtures was detected using cloud point measurements. In parallel with these measurements, the BisGMA/HEMA and adhesive/water mixtures were cast as films and polymerized. Molecular structure was recorded from the distinct features in the phase-separated adhesive using confocal Raman microspectroscopy (CRM). Human dentin specimens treated with Single Bond were analyzed with scanning electron microscopy (SEM) and CRM mapping across the dentin/adhesive interface. The model BisGMA/HEMA mixtures with ethanol and the commercial BisGMA-based adhesive experienced phase separation at approximately 25 vol% water. Raman spectra collected from the phase-separated adhesive indicated that the composition of the particles and surrounding matrix material was primarily BisGMA and HEMA, respectively. Based on SEM analysis, there was substantial porosity at the adhesive interface with dentin. Micro-Raman spectral analysis of the dentin/adhesive interface indicates that the contribution from the BisGMA component decreases by nearly 50% within the first micrometer. The morphologic results in corroboration with the spectroscopic data suggest that as a result of adhesive phase separation the hybrid layer is not an impervious 3-dimensional collagen/polymer network but a porous web characterized by hydrophobic BisGMA-rich particles distributed in a hydrophilic HEMA-rich matrix.

Bisphenol A-Glycidyl Methacrylate↗
PubMed1999

Application of a thermodynamic model to the prediction of phase separations in freeze-concentrated formulations for protein lyophilization.

Many of the compounds considered for use in pharmaceutical formulations demonstrate incompatibilities with other components at high enough concentrations, including pairs of polymers, polymers and salts, or even proteins in combination with polymers, salts, or other proteins. Freeze concentration can force solutions into a region where incompatibilities between solutes will manifest as the formation of multiple phases. Such phase separation complicates questions of the stability of the formulation as well as labile components, such as proteins. Yet, phase separation events are difficult to identify by common formulation screening methods. In this report, we use the osmotic virial expansion model of Edmond and Ogston (1) to describe phase-separating behavior of ternary aqueous polymer solutions. Second osmotic virial coefficients of polyethylene glycol 3350 (PEG) and dextran T500 were measured by light scattering. Assuming an equilibrium between ice and water in the freeze-concentrated solution, a degree of freeze concentration can be estimated, which, when combined with the phase separation spinodal, describes a "phase separation envelope" in which phase separation tendencies can be expected in the frozen solution. The phase separation envelope is bounded at low temperatures by the glass transition temperature of the freeze-concentrated solution. Scanning electron microscopic images and infrared spectroscopy of protein structure are provided as experimental evidence of the phase separation envelope in a freeze-dried system of PEG, dextran, and hemoglobin.

Dextrans↗
PubMed2003

Possibility of tissue separation caused by cell adhesion.

Adhesive molecules are suggested to play an important role when a single tissue is separated into two in developmental processes, illustrated by tissue-specific cadherins in the neural tube formation of amphibians. In this paper, we study the possibility for tissue separation to be carried out only by differential cell adhesion and random cell movement without any other morphogenetic mechanisms. We consider a two-dimensional regular triangular lattice filled with cells of three types (black, white, and gray). In the initial state, a cluster of black cells and a cluster of white cells are in contact and are surrounded by gray cells. Nearest-neighbor cells exchange their location at random, but the movement occurs faster if it increases the total adhesion. We considered separation to be successful if, in the final state, black cells and white cells kept their clusters but two clusters lost their direct contact with each other as gray cells are inserted between them. The maximum total adhesion (MTA) rule conjectures that the spatial pattern achieving maximum total adhesion might be that obtained in the final state. In the computer simulation, the runs for successful separation satisfied the condition predicted by the MTA rule. However, the condition for successful separation was more restricted than that predicted by the MTA rule. For some combinations of adhesions, it took an extremely long time to accomplish tissue separation. Finally, we discuss the role of homophilic adhesion molecules (such as cadherins) in the tissue separation processes, and show that the new expression of homophilic adhesion molecules cannot perform tissue separation without the change in other morphogenetic processes.

Animals↗
PubMed1984

Sequence of centromere separation another mechanism for the origin of nondisjunction.

The most commonly accepted view about the origin of aneuploidy is that it is due to errors in meiotic division. However, its rare occurrence makes it difficult to explain recurrent births of trisomic children to some parents. This problem causes more serious concern when one accepts that an abnormal (n + 1 or n - 1) sperm would enter fertilization by overriding thousands, or even millions, of normal haploid sperms. Also, the failure of aneuploidy to be induced in the offspring of mammals treated with mutagens raises questions about the effectiveness of the accepted mode of origin of errors. Current concepts also do not explain why one observes more errors of meiotic I, than of meiotic II, origin. It is known that most chromosomes separating at meta-anaphase junction in mitosis follow a nonrandom, genetically controlled sequence of separation. The present proposal makes use of out-of-phase separation of a rare chromosome, like premature separation in mitosis of the X in elderly humans or of an 18 in parents of trisomy 18 children. The suggestion is made that such out-of-phase separation results in aneuploid cell lines by total failure of the centromere to separate or by it separating too early, before the spindle is formed. The prematurely separating centromeres, it appears, do not attach to spindle fibers and hence cause nondisjunction. Such nondisjunction in embryonic stages will produce apparently normal individuals with mosaicism in somatic and/or gametic tissue. An individual carrying mosaicism in gonadal tissue will produce a large number of abnormal gametes, one of which may have a reasonable chance of entering fertilization. This mode of origin of aneuploidy takes care of all questions raised above and finds support in the data available in the literature. Several of the suggestions made in the hypothesis are easily testable.

Adult↗
PubMed1988

Separation of neutral lipid, free fatty acid and phospholipid classes by normal phase HPLC.

Normal phase high performance liquid chromatography methods are described for the separation of neutral lipid, fatty acid and five phospholipid classes using spectrophotometric detection at 206 nm. Separations were accomplished in less than 10 min for each lipid class. A mobile phase consisting of hexane/methyltertiarybutylether/acetic acid (100:5:0.02) proved effective in separating cholesteryl ester and triglyceride with recoveries of 100% for radiolabeled cholesteryl oleate and 98% for radiolabeled triolein. Free fatty acid and cholesterol were separated by two different mobile phases. The first, hexane/methyltertiarybutylether/acetic acid (70:30:0.02) effectively separated free fatty acids and cholesterol, but did not separate cholesterol from 1,2-diglyceride. A mobile phase consisting of hexane/isopropanol/acetic acid (100:2:0.02) effectively separated free fatty acid, cholesterol, 1,2-diglyceride and 1,3-diglyceride. Recoveries of oleic acid and cholesterol were 100% and 97%, respectively. Five phospholipid classes were separated using methyltertiarybutylether/methanol/aqueous ammonium acetate (pH 8.6) (5:8:2) as the mobile phase. The recoveries of phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine were each greater than 96%.

Animals↗
PubMed2004

Repeated maternal separation: differences in cocaine-induced behavioral sensitization in adult male and female mice.

RATIONALE: Repeated maternal separations profoundly alter the adult stress response, the development of the hypothalamic-pituitary-adrenal axis, and prominently, the GABAergic and monoaminergic systems. These neural changes are postulated to influence the vulnerability to drugs of abuse implicating glucocortocoids in the behavioral responses to psychomotor stimulants. OBJECTIVE: To investigate whether repeated brief maternal separation stress increases behavioral sensitization to cocaine in adult male and female mice, and to assess any concurrent changes in hippocampal glucocorticoid receptors and accumbal dopamine transporters. METHODS: Half of the litters were separated from the nest for 1 h/day from post-natal days 1 to 13. Starting on post-natal day 50, all mice were injected with either cocaine (10.0 mg/kg) or saline for 10 consecutive days. Locomotor activity was assessed in an open field on days 50, 54 and 59 via a tracking system. Approximately 10 and 40 days later, all mice were challenged with 7.5 mg/kg cocaine. RESULTS: Repeated maternal separation increased the hyperlocomotor response to 10.0 mg/kg cocaine regardless of gender. During expression tests (days 69/71, 99), male, but not female, mice with a history of maternal separation exhibited significant sensitized hyperactivity in response to cocaine. Male mice that were maternally separated and had no history of cocaine sensitization, demonstrated cross-sensitization to 7.5 mg/kg cocaine. Immunohistochemical analysis revealed that the hippocampal CA1 glucocorticoid receptor and nucleus accumbens dopamine transporter proteins were expressed more in females than in males, regardless of maternal separation experience. CONCLUSIONS: Repeated maternal separation is a stressor that can induce heightened sensitivity to low doses of cocaine, as expressed by hyperactivity. Furthermore, sex differences in glucocorticoid receptor and dopamine transporter expression may be responsible for the sexual dimorphic expression of behavioral sensitization to cocaine.

Animals↗
PubMed2004

Multidimensional LC-LC and LC-CE for high-resolution separations of biological molecules.

In multidimensional separations, two or more independent separation methods are coupled in an effort to resolve complex mixtures. The displacement mechanisms of each method should be orthogonal, such that little correlation exists between the retention of compounds in each dimension. When multiple orthogonal separation methods are coupled such that all sample components are subjected to complete analysis on all dimensions, the method is considered "comprehensive". The primary advantage of comprehensive multidimensional separations over their one-dimensional counterparts is the potential for dramatically enhanced resolution. High resolving power can be achieved because the peak capacity of a comprehensive multidimensional separation is roughly equal to the product of the individual peak capacities of each dimension. In this review, the theory and instrumentation of two-dimensional liquid chromatography (LC-LC) and liquid chromatography-capillary electrophoresis (LC-CE) separations are discussed. Some applications of these techniques to the separation of biological molecules are highlighted. Future directions for the development of multidimensional separations are also considered.

Chromatography, Liquid↗
PubMed2004

Craniopagus twins: embryology, classification, surgical anatomy, and separation.

INTRODUCTION: With recent advances in brain imaging and neurosurgical techniques, there has been a renewed interest in the surgical separation of craniopagus twins. Successful separation in recent cases, along with widespread publicity, has attracted craniopagus twins from all over the world to be referred to pediatric neurosurgical centers for evaluation and consideration for surgical separation. SEPARATION OF BLOOD SUPPLY: It has become apparent, however, that the most critical decisions in surgical planning are related to separation of the blood supply to the conjoined brains. In fact, in craniopagus twins that survive pregnancy or the first few days of life, there is usually little shared brain tissue. The shared blood supply is far and away the more critical issue. It is very difficult to successfully separate craniopagus twins in one surgical procedure. Staged separation, with gradual re-routing of the shared blood supply, has been a successful alternative. CASE STUDIES AND DISCUSSION: We discuss here our experience with three sets of craniopagus twins and our approach to staged separation.

Abnormalities, Multiple↗