Formation Of Visible Aggregates Research Articles

Formation of Visible Aggregates between Rolling Circle Amplification Products and Magnetic Nanoparticles as a Strategy for Point-of-Care Diagnostics.

Visual detection of rolling circle amplification products (RCPs) has been achieved by specific aggregation with magnetic nanoparticles. The method presented here reliably generates aggregates in 1.5 h; these are visible to the naked eye in samples containing at least 0.4 fmol of RCPs. In addition, alternate current susceptometry and absorbance spectroscopy have also been used to quantify the amplified products. The specificity of the detection method was tested, and no non-specific aggregation was detected in samples containing up to 20 fmol of non-complementary amplified DNA. This method is a versatile tool for detecting pathogenic DNA in point-of-care diagnostics, with no readout equipment required. However, chips and automated assays can be used in conjugation with the developed method since detection and quantification can be achieved by commercially available readout instruments.

Xanthan gum − mucin complexation: Molecular interactions, thermodynamics, and rheological analysis

Texture perception, astringency phenomena, and oral sensation are directly influenced by molecular interactions. This study systematically characterizes the diverse array of molecular interactions in a binary model system comprising of mucin, the major viscosity enhancer of biological fluids, and xanthan gum, a major food hydrocolloid, at a pH range of 1–7. Coexistence of xanthan gum and mucin at 1:2 ratio (w/w) under acidic pH results in phase separation, as evidenced by the formation of visible aggregates. ζ-Potential data rule out any relevant electrostatic interactions. Fluorimetry analysis points to the existence of two distant binding regimes of mucin, manifesting at low (y1) and high (y2) xanthan gum concentrations, corroborating a pH-independent two-step binding mechanism. Enthalpy-dominated (ΔH° < 0) interaction occur at pH 7, whilst, entropy-driven (ΔS° > 0) interactions (classical hydrophobic forces) stabilize both transient and static complexes at pH 3. Both macromolecules interact spontaneously (ΔG° < 0) at the two pH values. Based on rheological data, the macromolecular interaction proves to be less dependent on the mucin to xanthan gum weight ratios. Nevertheless, the partial substitution of mucin with xanthan gum leads to enhanced viscoelasticity (G' > G") and increased relaxation times (λ). Furthermore, the xanthan gum inclusion in mucin systems at a 5:5 (w/w) ratio elevates the apparent viscosity (η) ≥ 45-fold (21.59 Pa s) and ≥70-fold (33.52 Pa s) in comparison with mucin (0.46 − 0.48 Pa s) at pH 3 and pH 7, respectively. The findings of this study highlight the physicochemical basis of designing dysphagia diets, modulating food functionality, and tailoring the organoleptic properties of food systems.

Heterotrophic Bacteria Enhance the Aggregation of the Marine Picocyanobacteria Prochlorococcus and Synechococcus.

Marine picocyanobacteria are ubiquitous primary producers across the world’s oceans, and play a key role in the global carbon cycle. Recent evidence stemming from in situ investigations have shown that picocyanobacteria are able to sink out of the euphotic zone to depth, which has traditionally been associated with larger, mineral ballasted cells. The mechanisms behind the sinking of picocyanobacteria remain a point of contention, given that they are too small to sink on their own. To gain a mechanistic understanding of the potential role of picocyanobacteria in carbon export, we tested their ability to form “suspended” (5–60 μm) and “visible” (ca. > 0.1 mm) aggregates, as well as their production of transparent exopolymer particles (TEP)—which are a key component in the formation of marine aggregates. Additionally, we investigated if interactions with heterotrophic bacteria play a role in TEP production and aggregation in Prochlorococcus and Synechococcus by comparing xenic and axenic cultures. We observed TEP production and aggregation in batch cultures of axenic Synechococcus, but not in axenic Prochlorococcus. Heterotrophic bacteria enhanced TEP production as well as suspended and visible aggregate formation in Prochlorococcus, while in Synechococcus, aggregation was enhanced with no changes in TEP. Aggregation experiments using a natural plankton community dominated by picocyanobacteria resulted in aggregation only in the presence of the ballasting mineral kaolinite, and only when Synechococcus were in their highest seasonal abundance. Our results point to a different export potential between the two picocyanobacteria, which may be mediated by interactions with heterotrophic bacteria and presence of ballasting minerals. Further studies are needed to clarify the mechanistic role of bacteria in TEP production and aggregation of these picocyanobacteria.

A thiol probe for measuring unfolded protein load and proteostasis in cells

When proteostasis becomes unbalanced, unfolded proteins can accumulate and aggregate. Here we report that the dye, tetraphenylethene maleimide (TPE-MI) can be used to measure cellular unfolded protein load. TPE-MI fluorescence is activated upon labelling free cysteine thiols, normally buried in the core of globular proteins that are exposed upon unfolding. Crucially TPE-MI does not become fluorescent when conjugated to soluble glutathione. We find that TPE-MI fluorescence is enhanced upon reaction with cellular proteomes under conditions promoting accumulation of unfolded proteins. TPE-MI reactivity can be used to track which proteins expose more cysteine residues under stress through proteomic analysis. We show that TPE-MI can report imbalances in proteostasis in induced pluripotent stem cell models of Huntington disease, as well as cells transfected with mutant Huntington exon 1 before the formation of visible aggregates. TPE-MI also detects protein damage following dihydroartemisinin treatment of the malaria parasites Plasmodium falciparum. TPE-MI therefore holds promise as a tool to probe proteostasis mechanisms in disease.

Polyalanine expansions drive a shift into α-helical clusters without amyloid-fibril formation.

Polyglutamine (polyGln) expansions in nine human proteins result in neurological diseases and induce the proteins' tendency to form β-rich amyloid fibrils and intracellular deposits. Less well known are at least nine other human diseases caused by polyalanine (polyAla)-expansion mutations in different proteins. The mechanisms of how polyAla aggregates under physiological conditions remain unclear and controversial. We show here that aggregation of polyAla is mechanistically dissimilar to that of polyGln and hence does not exhibit amyloid kinetics. PolyAla assembled spontaneously into α-helical clusters with diverse oligomeric states. Such clustering was pervasive in cells irrespective of visible aggregate formation, and it disrupted the normal physiological oligomeric state of two human proteins natively containing polyAla: ARX and SOX3. This self-assembly pattern indicates that polyAla expansions chronically disrupt protein behavior by imposing a deranged oligomeric status.

Control of heat treatment and storage temperature prevents the formation of visible aggregates in acidic whey dispersions over a 6-month storage period

Acidic whey protein isolate (WPI) dispersions are widely applied for translucent whey-based beverages. However, developing beverages that maintain clarity during extended storage can be a challenge. The formation of visible aggregates occurs recurrently during storage, leading to reduced consumer satisfaction. Batch-to-batch variation with respect to the degree of aggregation also is observed, which impairs shelf-life determination. In this study, two WPI batches (A and B) with different levels of visible aggregate formation during the storage of acidic dispersions were analyzed. Visual evaluations, oscillatory rheology, and absorbance measurements (A500) were applied to assess the formation of visible aggregates in 70 g protein L−1 WPI dispersions during a 6-month period. It was shown, that by changing the heat treatment from the conventional conditions of 95 °C for 180 s to 120 °C for 20 s and/or lowering the storage temperature from the conventional 20 °C–4 °C, the formation of visible aggregates was prevented over the 6-month storage period. The protein composition of the aggregates and the surrounding clear liquid was analyzed by 2-dimensional gel-electrophoreses. The visible aggregate was highly enriched by a low pI spot, possibly containing caseinomacropeptide and/or proteolytically truncated fragments of β-lactoglobulin and α-lactalbumin.

Understanding the relevance of local conformational stability and dynamics to the aggregation propensity of an IgG1 and IgG2 monoclonal antibodies

Aggregation of monoclonal antibodies is often a multi-step process involving structural alterations in monomeric proteins and subsequent formation of soluble or insoluble oligomers. The role of local conformational stability and dynamics of native and/or partially altered structures in determining the aggregation propensity of monoclonal antibodies, however, is not well understood. Here, we investigate the role of conformational stability and dynamics of regions with distinct solvent exposure in determining the aggregation propensity of an IgG1 and IgG2 monoclonal antibody. The temperatures employed span the pre-unfolding range (10-40°C) and the onset temperatures (T onset ) for exposure of apolar residues (≈ 50°C), alterations in secondary structures (≈ 60°C) and initiation of visible aggregate formation (≈ 60°C). Solvent-exposed regions were found to precede solvent-shielded regions in an initiation of aggregation for both proteins. Such a process was observed upon alterations in overall tertiary structure while retaining the secondary structures in both the proteins. In addition, a greater dynamic nature of solvent-shielded regions in potential intermediates of IgG1 and the improved conformational stability increased its resistance to aggregation when compared to IgG2. These results suggest that local conformational stability and fluctuations of partially altered structures can influence the aggregation propensity of immunoglobulins.

The effect of commercial sterilization regimens on micellar casein concentrates

This work focused on evaluating the effects of UHT sterilization and in-container retorting on the stability and physical properties of micellar casein concentrates (MCC). The study was performed on MCC obtained by membrane separation, with casein concentrations between 5 and 10%. The UHT and retorting regimens were designed to achieve the same microbial inactivation effect. Ultra-high temperature treatment was performed in a pilot-scale MicroThermics heating system (MicroThermics Inc., Raleigh, NC), and retorting in an FMC multipurpose laboratory retort (Steritort; FMC Corp., San Jose, CA). The heat-treated and the non-heat-treated MCC controls were evaluated for pH, mineral profile, ζ-potential, particle size, and rheological properties for up to 24h after heat treatment. The treatments were performed in triplicate, and differences among samples were evaluated using statistical analyses. Retorting resulted in slight aggregation in the MCC, whereas UHT caused the formation of visible aggregates and coagulation. The UHT-treated MCC had higher viscosity than retorted MCC, and displayed predominantly solid-like rheological behavior, indicative of structure formation. These effects were, at least in part, attributed to a change in mineral equilibrium, which affected the stability of the casein micelles, but additional mechanisms such as κ-casein dissociation may also play a significant role in these heat-induced changes. Drying of MCC accentuated the observed instabilities, as dried and reconstituted micellar casein concentrates (R-MCC) were more unstable to UHT sterilization than the MCC that had not undergone drying. The results of this study provide valuable information about the sterilization behavior and physical properties of MCC, which can be useful to processors in the development and manufacture of shelf-stable casein-based products and beverages.

Superoxide dismutase 1 mutants related to amyotrophic lateral sclerosis induce endoplasmic stress in neuro2a cells

One of the common features of damaged neurons in many neurodegenerative diseases is the presence of abnormal aggregates of the disease-related proteins. In amyotrophic lateral sclerosis (ALS) of both sporadic and familial forms, protein aggregates are found in the affected spinal cords. In familial ALS with mutations in copper-zinc superoxide dismutase 1 (SOD1), the propensity of SOD1 for aggregation is known to increase with the mutation. In the present study, we examined whether the aggregate-prone SOD1 mutants induce endoplasmic reticulum (ER) stress and the inhibition of the ER stress protects the cells. The ALS-related mutant G85R SOD1 and G93A SOD1 formed visible aggregates and caused cell death possibly by apoptosis when over-expressed in neuro2a cells. Interestingly, the rate of the mutant SOD1-induced cell death was greater than that of the visible aggregate formation. Expression of the mutant SOD1 caused signs of both early and late ER stress responses, namely, RNA-dependent protein kinase-like ER kinase and eukaryotic initiation factor alpha phosphorylation, Jun amino-terminal kinase activation, activating transcription factor 6-translocation, X-box binding protein 1 mRNA splicing, and caspase 12 activation. The X-box binding protein 1 mRNA splicing activation was also detected in the mutant SOD1-expressing cells even without the visible aggregates. The cell death induced by the mutant SOD1 over-expression looked like apoptosis as evidenced by nuclear morphology and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling. Importantly, an ER stress inhibitor, salubrinal delayed the formation of insoluble aggregates of the mutant SOD1 and suppressed the mutant-induced cell death. In addition, over-expression of the ER-targeted Bcl-xL protected the cells from the mutant SOD1-induced cytotoxicity. These results suggest that the misfolding of ALS-related mutant SOD1 induces ER stress possibly prior to the formation of visible aggregates, which may contribute to the motor neuron degeneration in ALS pathogenesis.

Time-lapse analysis of aggregate formation in an inducible PC12 cell model of Huntington's disease reveals time-dependent aggregate formation that transiently delays cell death

Huntington's disease (HD) is a progressive neurodegenerative disease caused by a CAG repeat expansion in the coding region of the HD gene. The translated polyglutamine expansion causes the formation of insoluble aggregates in the brains of HD patients and transgenic mouse models. However, the relationship between aggregate formation and neuropathology remains unknown. We used fluorescent protein tagging and live-cell time-lapse microscopy to study visible aggregate formation and its relationship to cell death in transgenic PC12 cells. We used cell lines expressing a fragment of huntingtin exon 1 with either 23 (wild type) or 74 (mutant) glutamines fused to enhanced green fluorescent protein under the control of an inducible promoter. Live cells were observed in real time after transgene induction for up to 96 h. We found that aggregate formation was time-dependent and predominantly nuclear in these cells. We followed inclusion formation in individual cells, examining the cells every 10 min for up to 48 h. This revealed new details of inclusion formation. Initial aggregate formation was rapid (often <1 h), but many (18–>48) h were needed to establish a final aggregate phenotype. Aggregates formed in a dynamic manner and were in constant motion within cell nuclei throughout their maturation. The formation of large aggregates occurred more frequently in cells that survived longer. However, aggregate size was not a good predictor of cell death, since cells could die with either large (>2 μm), small (<0.5 μm) or no visible aggregates. Cells that formed large aggregates survived longer than cells that formed small aggregates or no aggregates at all. However, the time taken for a cell to die decreased as a function of increasing size of final aggregate formed. Further, cells that formed aggregates earlier tended to die earlier. Together our data are compatible with a toxic role for aggregates/aggregation and support the ‘toxic precursor’ hypothesis. However, they also suggest that at some stages, the process of aggregate formation is cytoprotective.

Self-Assembly of Recombinant Amphiphilic Oligopeptides into Vesicles

The aim of the present study was to design amphiphilic oligopeptides that can self-assemble into vesicular structures. The ratio of hydrophilic to hydrophobic block length was varied, and peptides were designed to have a hydrophobic tail in which the bulkiness of the amino acid side groups increases toward the hydrophilic domain (Ac-Ala-Ala-Val-Val-Leu-Leu-Leu-Trp-Glu(2/7)-COOH). These peptides were recombinantly produced in bacteria as an alternative to solid-phase synthesis. We demonstrate with different complementary techniques (dynamic and static light scattering, tryptophan fluorescence anisotropy, and electron microscopy) that these amphiphilic peptides spontaneously form vesicles with a radius of approximately 60 nm and a low polydispersity when dispersed in aqueous solution at neutral pH. Morphology and size of the vesicles were relatively insensitive to the variations in hydrophilic block length. Exposure to acidic pH resulted in formation of visible aggregates, which could be fully reversed to vesicles upon pH neutralization. In addition, it was demonstrated that water-soluble molecules can be entrapped inside these peptide vesicles. Such peptide vesicles may find applications as biodegradable drug delivery systems with a pH-dependent release profile.

The inflammation-sensitive protein alpha 1-anti-chymotrypsin neutralizes fibrillar aggregation and cytotoxicity of the beta-amyloid peptide more effectively than alpha 1-antitrypsin

Objectives: A neuroinflammatory process, triggered by amyloid-beta (Aβ)-peptide, is thought to play a central role in the neurodegenerative process leading to Alzheimer's disease (AD). Aβ 25–35 retains the functionality of Aβ 42 and was employed to investigate the effects of inflammation-sensitive proteins (ISPs) α1-antichymotrypsin (A1ACT) and α1-antitrypsin (A1AT) on fibrillar aggregation and cytotoxicity. Design and methods: Inhibitory concentrations of the ISPs were determined in an established human red blood cell lysis model of Aβ-cytotoxicity. For studies of Aβ-fibrillar aggregation CSF levels of A1ACT (0.041 μM)/A1AT (0.11 μM) were incubated with Congo Red dye 25 μM + Aβ 25–35 10 μM noting the formation of visible aggregates and spectrophotometric changes over 24 h. Results: A1ACT at CSF reported levels inhibited fibrillar aggregation and cytotoxicity while A1AT at CSF reported levels failed to cause a similar inhibition. Conclusions: A1ACT neutralizes fibrillar aggregation and cytotoxicity of Aβ-peptide more effectively than A1AT. Both proteins are known to be co-deposited with Aβ within senile plaques of AD brains.

The first 17 amino acids of Huntingtin modulate its sub-cellular localization, aggregation and effects on calcium homeostasis

A truncated form of the Huntington's disease (HD) protein that contains the polyglutamine repeat, Httex1p, causes HD-like phenotypes in multiple model organisms. Molecular signatures of pathogenesis appear to involve distinct domains within this polypeptide. We studied the contribution of each domain, singly or in combination, to sub-cellular localization, aggregation and intracellular Ca2+ ([Ca2+]i) dynamics in cells. We demonstrate that sub-cellular localization is most strongly influenced by the first 17 amino acids, with this sequence critically controlling Httex1p mitochondrial localization and also promoting association with the endoplasmic reticulum (ER) and Golgi. This domain also enhances the formation of visible aggregates and together with the expanded polyQ repeat acutely disrupts [Ca2+]i levels in glutamate-challenged PC12 cells. Isolated cortical mitochondria incubated with Httex1p resulted in uncoupling and depolarization of these organelles, further supporting the idea that Httex1p-dependent mitochondrial dysfunction could be instrumental in promoting acute Ca2+ dyshomeostasis. Interestingly, neither mitochondrial nor ER associations seem to be required to promote long-term [Ca2+]i dyshomeostasis.

Tumor suppressor PTEN affects tau phosphorylation: deficiency in the phosphatase activity of PTEN increases aggregation of an FTDP-17 mutant Tau

BackgroundAberrant hyperphosphorylation of tau protein has been implicated in a variety of neurodegenerative disorders. Although a number of protein kinases have been shown to phosphorylate tau in vitro and in vivo, the molecular mechanisms by which tau phosphorylation is regulated pathophysiologically are largely unknown. Recently, a growing body of evidence suggests a link between tau phosphorylation and PI3K signaling. In this study, phosphorylation, aggregation and binding to the microtubule of a mutant frontal temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) tau in the presence of tumor suppressor PTEN, a major regulatory component in PI3K signaling, were investigated.ResultsPhosphorylation of the human mutant FTDP-17 tau, T40RW, was evaluated using different phospho-tau specific antibodies in the presence of human wild-type or phosphatase activity null mutant PTEN. Among the evaluated phosphorylation sites, the levels of Ser214 and Thr212 phospho-tau proteins were significantly decreased in the presence of wild-type PTEN, and significantly increased when the phosphatase activity null mutant PTEN was ectopically expressed. Fractionation of the mutant tau transfected cells revealed a significantly increased level of soluble tau in cytosol when wild-type PTEN was expressed, and an elevated level of SDS-soluble tau aggregates in the presence of the mutant PTEN. In addition, the filter/trap assays detected more SDS-insoluble mutant tau aggregates in the cells overexpressing the mutant PTEN compared to those in the cells overexpressing wild-type PTEN and control DNA. This notion was confirmed by the immunocytochemical experiment which demonstrated that the overexpression of the phosphatase activity null mutant PTEN caused the mutant tau to form aggregates in the COS-7 cells.ConclusionTumor suppressor PTEN can alleviate the phosporylation of the mutant FTDP-17 tau at specific sites, and the phosphatase activity null PTEN increases the mutant tau phosphorylation at these sites. The changes of the tau phosphorylation status by ectopic expression of PTEN correlate to the alteration of the mutant tau's cellular distribution. In addition, the overexpression of the mutant PTEN can increase the level of the mutant tau aggregates and lead to the formation of visible aggregates in the cells.

Protein FOG - a streptococcal inhibitor of neutrophil function

Several strains of group G streptococci (GGS) form aggregates when grown in vitro. Aggregating strains interact with fibrinogen, and this study reports the isolation of a novel self-associating and fibrinogen-binding protein of GGS, denoted protein FOG. Sequencing of the fog gene revealed structural similarity with M proteins of both GGS and group A streptococci (GAS). Analogous to GAS, GGS were found to multiply in human blood. All strains of GGS express protein G, a protein known to interact with the constant region of immunoglobulin G and albumin. Surprisingly, a clinical isolate expressing protein G, but lacking protein FOG, was killed in human whole blood; however, the addition of intact soluble protein FOG restored the ability of the bacteria to survive and multiply in human blood. This is believed to be the first report of a soluble M-like protein salvaging an M-negative strain from being killed. The antibactericidal property of protein FOG is dependent on its fibrinogen-binding activity. Thus, in plasma, FOG precipitates fibrinogen, and when added to whole blood, protein FOG triggers the formation of visible aggregates comprising fibrinogen and neutrophils that are disabled in their killing of the bacteria. Moreover, the results emphasize the importance of an intact FOG molecule, as presented on the bacterial surface, for full protective effect.

Interactive aggregation and sedimentation of diatoms and clay‐sized lithogenic material

Monospecific diatom cultures (Thalassiosira punctigera and Skeletonema costatum) were incubated in rotating cylinders together with clay suspensions, present in a range of concentrations (5–100 mg kaolinite L−1) and as different minerals (50 mg L−1 kaolinite, smectite, illite, and clayȁ;sized quartz powder). The addition of lithogenic suspensions to diatom cultures accelerated the formation of visible aggregates in the roller tanks by a factor of &lt;3. Aggregate size decreased and density increased proportionally to the amount of kaolinite added to the diatom cultures. In the presence of kaolinite and illite, aggregate sizes were smaller and sinking rates lower than in the presence of smectite and quartz. The influence of lithogenic matter on the sinking velocities of aggregates was ambiguous. Compound aggregates sank faster with increasing amounts of lithogenic matter present in cultures of T. punctigera until a certain ratio between lithogenic and biogenic material was reached; further increasing the amount of lithogenic matter did not increase sinking rates significantly. In contrast, increasing the concentration of kaolinite added to cultures of S. costatum could decrease sinking velocities of the evolving compound aggregates. This nonlinear behavior is argued to be primarily a function of aggregate composition on aggregate sizes and excess densities. Although the possibility of a mutual acceleration of vertical flux of algae and clay is confirmed, the results show that the presence of lithogenic material could also decrease the downward flux of phytoplankton biomass.

Polyglutamine Expansion, Protein Aggregation, Proteasome Activity, and Neural Survival

Huntington's disease (HD) is one of eight established triplet repeat neurodegenerative disorders, which are collectively caused by the genetic expansion of polyglutamine repeats. While the mechanism(s) by which polyglutamine expansion causes neurodegeneration in each of these disorders is being intensely investigated, the underlying cause of polyglutamine toxicity has not been fully elucidated. A number of studies have focused on the potential role of protein aggregation and disruption of the proteasome proteolytic pathway in polyglutamine-mediated neurodegeneration. However, at present it is not clear whether polyglutamine-mediated protein aggregation is sufficient to induce cell death, nor has it been clearly determined whether proteasome inhibition precedes, coincides, or occurs as the result of the formation of polyglutamine-associated protein aggregation. To address these important components of polyglutamine toxicity, in the present study we utilized neural SH-SY5Y cells stably transfected with polyglutamine-green fluorescent protein constructs to examine the effects of polyglutamine expansion on protein aggregation, proteasome activity, and neural cell survival. Data from the present study demonstrate that polyglutamine expansion does not dramatically impair proteasome activity or elevate protein aggregate formation under basal conditions, but does significantly impair the ability of the proteasome to respond to stress, and increases stress-induced protein aggregation following stress, all in the absence of neural cell death.

Evaluation of compatibility of methacrylic copolymers by capillary viscometry

The estimation of the compatibility of different pairs of polymers can be based on capillary viscometry data for ternary polymer-polymer-solvent systems using mathematical models based on the slope of the Huggins equation (Δb) and Huggins constant (Δk′). In this study, the compatibility of binary mixtures of six types of methacrylic copolymers with similar molecular weights but different functional groups [one characterized by amine groups (EuE), two by ammonium groups (EuRL EuRS), two by carboxylic groups (EuL EuS), and one without charge (EuNE)] was evaluated using these methods. On the basis of Huggins and Kraemer constants, acetone and tetrahydrofurane were selected as good solvents for the programmed blends. Cationic copolymers mixed with anionic copolymers showed the formation of visible aggregates. The study performed on the other blends showed that EuRL and EuRS could be considered compatible with EuE; EuNE was incompatible with both EuL and EuE, EuL and EuS were incompatible between them. EuRL and EuRS could be considered compatible even if the weight ratio seems to influence the behavior of the two copolymers. The Δk′ approach seems to be more robust than the Δb model. The compatibility of different pairs of methacrylic copolymers with similar molecular weights could be evaluated using capillary viscometry. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 76: 1662–1668, 2000

Mechanism of Freeze–Thaw Instability of Aluminum Hydroxycarbonate and Magnesium Hydroxide Gels

□ The effect of freeze-thaw cycles on the physical stability of aluminum hydroxycarbonate and magnesium hydroxide gels was studied. Coagulation following a freeze-thaw cycle, leading to the formation of visible aggregates, affected the content uniformity of both gels. The freeze-thaw cycles did not affect the crystal form or surface characteristics of the gels as determined by X-ray powder diffraction and point of zero charge, but caused a slight reduction in the rate of acid neutralization and a large increase in the rate of sedimentation. The greatest effect was observed after the first freeze-thaw cycle. While the duration of freezing was not a factor, the rate of freezing was important and was inversely related to the aggregate size. The aggregates which formed following a freeze-thaw cycle were not redispersed by shaking, but were reversed by ultrasonic treatment or homogenization. The adsorption of polymers or surface-active agents prior to freezing reduced and, in some cases, prevented the formation of aggregates. The physical instability produced by a freeze-thaw cycle was explained by the modified DLVO theory. The force exerted on the particles by the growing ice crystals forced the particles into the primary minimum, producing strong interparticle attraction. On thawing, simple agitation did not provide enough force to overcome the attractive force of the primary minimum. Adsorption of polymers or surface-active agents increased the steric repulsive force and prevented the particles from reaching the primary minimum.

Platelet aggregation in laminar flow. I. Adenosine diphosphate concentration, time and shear rate dependence

The relative contributions of physiologic activators and shear rates (G) on the formation and stability of platelet aggregates for in vitro laminar flow need to be characterized for conditions of low G believed to be important in vivo . Platelet aggregation was therefore studied in human citrated platelet-rich plasma (PRP) prepared at 37°C for G = 0–190 sec −1 in laminar flow in a cone-in-plate device as a function of adenosine diphosphate (ADP) activation. Macroaggregation (TA) was assessed by visual observation and light transmission (%T), as routinely done in aggregometry, while actual percent aggregation (PA) was determined by microscopy of subsamples from the decrease in single platelet concentration. Unactivated PRP never yielded shear-induced aggregation while samples pre-mixed with low [ADP] (≲ 0.7 μM) led to large visible aggregate formation stable only for G ≲ 30–60 sec −1, apparently arising from pre-existing microaggregates. Direct ADP injections into the flow device showed that at low [ADP] (< 1 μM), maximal values for both PA and TA were reached when step-wise increases in G reached ≲ 30 sec −1, with TA decreasing at higher G; these values were only some 10–20% of maximal values achieved with high [ADP] (≳ 4 μM), when PA was again maximal at G ≳ 30 sec −1, but TA expressed as rate of aggregation continued to increase with G up to 150 sec −1. The results are discussed in terms of the temporal shear-rate and ADP-dependent dynamics of a platelet stickiness factor, ‘s’.