Preparation Of High Molecular Weight Research Articles

Tau seeding and spreading in vivo is supported by both AD-derived fibrillar and oligomeric tau

Insoluble fibrillar tau, the primary constituent of neurofibrillary tangles, has traditionally been thought to be the biologically active, toxic form of tau mediating neurodegeneration in Alzheimer’s disease. More recent studies have implicated soluble oligomeric tau species, referred to as high molecular weight (HMW), due to their properties on size-exclusion chromatography, in tau propagation across neural systems. These two forms of tau have never been directly compared. We prepared sarkosyl-insoluble and HMW tau from the frontal cortex of Alzheimer patients and compared their properties using a variety of biophysical and bioactivity assays. Sarkosyl-insoluble fibrillar tau comprises abundant paired-helical filaments (PHF) as quantified by electron microscopy (EM) and is more resistant to proteinase K, compared to HMW tau, which is mostly in an oligomeric form. Sarkosyl-insoluble and HMW tau are nearly equivalent in potency in HEK cell bioactivity assay for seeding aggregates, and their injection reveals similar local uptake into hippocampal neurons in PS19 Tau transgenic mice. However, the HMW preparation appears to be far more potent in inducing a glial response including Clec7a-positive rod microglia in the absence of neurodegeneration or synapse loss and promotes more rapid propagation of misfolded tau to distal, anatomically connected regions, such as entorhinal and perirhinal cortices. These data suggest that soluble HMW tau has similar properties to fibrillar sarkosyl-insoluble tau with regard to tau seeding potential, but may be equal or even more bioactive with respect to propagation across neural systems and activation of glial responses, both relevant to tau-related Alzheimer phenotypes.

Role and Effectiveness of Intra-articular Injection of Hyaluronic Acid in the Treatment of Knee Osteoarthritis: A Systematic Review.

Osteoarthritis (OA) is a degenerative joint disease that causes persistent joint pain and stiffness of mainly the large peripheral weight-bearing joints. It is a leading cause of functional disability and poor quality of life. Various modalities of therapy are recommended by different research organizations at different stages of OA including non-pharmacological, pharmacological, and surgical interventions. Intra-articular injections of hyaluronic acid (HA) is widely used for over three decades in the treatment of OA. However controversies exist regarding its safety and efficacy, the number of injections and courses, type of preparation, duration of its effects, and combining it with other drugs or molecules. This study aimed to review the most recent data available in the published literature to address these.Electronic databases like Medline, Embase, ProQuest, and Google Scholar were searched for articles using keywords, intraarticular injections, hyaluronic acid, and osteoarthritis knee. The review was carried out as per PRISMA guidelines.Thirty-eight randomized control trials (RCTs) investigating the efficacy and safety of intra-articular injection of HA were included in the systematic review. Out of the 38 studies, 22 (57.9%) were double-blind, eight (21%) single-blind, three (7.9%) non-blind, four (10%) with simple randomization, and one (2.7%) was open-labeled. Total 5,025 patients were included in these studies. The mean age of the patients was 60.28 years and the osteoarthritis grade of the knee joint was 1 to 3. HA was studied as a test preparation in 19 (50%) while in another 19 (50%) it was studied as a control. In 24 (63.2%) studies, HA was used as high molecular weight preparation in eight (21%) as low molecular weight preparation while in six studies the information was not available. HA was used as a standalone preparation in 31 studies, in two studies it was injected with platelet-rich plasma (PRP) and with either low-level laser therapy (LLLT), triamcinolone (TA), betamethasone (CS), poly deoxyribonucleotide (PDRN) or dexamethasone (DX) in one study each. In the majority of the studies, HA was given as a single injection (52.6% studies) or weekly three injections (28.9% studies). In 13.2 %, it was given as weekly 5 injections and in 5.3% as weekly two injections.IA-HA injections have a limited role in the treatment of knee osteoarthritis in those patients who do not have sufficient pain relief with topical or oral medication and physical therapy. It is safe and effective except for minor side effects such as local pain and swelling lasting for a few days. Severe allergic reactions are extremely rare. They provide adequate pain relief and functional improvement for up to six months irrespective of a number of injections and type of preparations used. The combination formulations with corticosteroids or PRP or MSCs show better results than HA alone. Combining HA with newer molecules such as peptides or diclofenac for sustained and disease-modifying effects requires more studies in the future.

Hydrogel droplet single-cell processing: DNA purification, handling, release, and on-chip linearization.

The preparation and handling of mammalian single-cell genomic DNA is limited by the complexity bottleneck inherent to performing multi-step, multi-reagent operations in a microfluidic environment. We have developed a method for benchtop preparation of high-molecular weight, intact, single-cell genomes and demonstrate the extraction of long nucleic acid molecules in a microfluidic system. Lymphoblasts are encapsulated inside of alginate microparticles using a droplet microfluidics, and cells are lysed in bulk. The purified genomes are then delivered to and imaged on a dedicated microfluidic device. High-molecular weight DNA is protected from shear and retains its original cellular identity. Using this encapsulation protocol, we were able to extract individual nucleic acid strands on the millimeter scale inside of a microfluidic channel.

Molecular fingerprinting of complex grass allergoids: size assessments reveal new insights in epitope repertoires and functional capacities

Background Subcutaneous allergen immunotherapy (SCIT) is a well-documented treatment for allergic disease which involves injections of native allergen or modified (allergoid) extracts. The use of allergoid vaccines is a growing sector of the allergy immunotherapy market, associated with shorter-course therapy. The aim of this study was the structural and immunological characterisation of group 1 (Lol p 1) IgG-binding epitopes within a complex mix grass allergoid formulation containing rye grass.Methods HP-SEC was used to resolve a mix grass allergoid preparation of high molecular weight into several distinct fractions with defined molecular weight and elution profiles. Allergen verification of the HP-SEC allergoid fractions was confirmed by mass spectrometry analysis. IgE and IgG immunoreactivity of the allergoid preparations was explored and Lol p 1 specific IgG-binding epitopes mapped by SPOT synthesis technology (PepSpot™) with structural analysis based on a Lol p 1 homology model.Results Grass specific IgE reactivity of the mix grass modified extract (allergoid) was diminished in comparison with the mix grass native extract. A difference in IgG profiles was observed between an intact mix grass allergoid preparation and HP-SEC allergoid fractions, which indicated enhancement of accessible reactive IgG epitopes across size distribution profiles of the mix grass allergoid formulation. Detailed analysis of the epitope specificity showed retention of six Lol p 1 IgG-binding epitopes in the mix grass modified extract.Conclusion The structural and immunological changes which take place following the grass allergen modification process was further unravelled revealing distinct IgG immunological profiles. All epitopes were mapped on the solvent exposed area of Lol p 1 homology model accessible for IgG binding. One of the epitopes was identified as an ‘immunodominant’ Lol p 1 IgG-binding epitope (62-IFKDGRGCGSCFEIK-76) and classified as a novel epitope. The results from this study support the concept that modification allows shorter-course therapy options as a result of providing an IgG epitope repertoire important for efficacy. Additionally, the work paves the way to help further develop methods for standardising allergoid platforms.

Styrene‐vinyl pyridine diblock copolymers: Achieving high molecular weights by the combination of anionic and reversible addition–fragmentation chain transfer polymerizations

AbstractAnionic and reversible addition–fragmentation chain transfer (RAFT) polymerizations were combined for the preparation of high molecular weight (MW) amphiphilic diblock copolymers based on the hydrophobic styrene (Sty) and the more polar 2‐vinyl pyridine (2VPy) or 4‐vinyl pyridine (4VPy). In particular, four amphiphilic Sty‐VPy diblock copolymers with MWs up to 271,000 g mol–1were prepared. For the polymer synthesis, first, living anionic polymerization of Sty usingsec‐butyl‐lithium as initiator in tetrahydrofuran at −70 °C, followed by termination with ethylene oxide were employed for the preparation of OH‐functionalized homopolyStys. Subsequently, a modification of the OH‐terminal group was performed by the attachment of a 4‐cyanopentanoic acid dithiobenzoate chain transfer agent (CTA) group, giving a polySty macroRAFT CTA, which was extended with 2VPy or 4VPy units using RAFT polymerization. Thus, the prepared diblock copolymers comprised a first block which was near‐monodisperse in size, and a second more heterogeneous block. All diblock copolymers were characterized in terms of their MWs and compositions by gel permeation chromatography and1H NMR spectroscopy, respectively, giving results close to the theoretically expected values. Films cast from chloroform solutions of the diblock copolymers were investigated in terms of their bulk morphologies using transmission electron microscopy, which indicated that the minority block consistently formed the discontinuous microphase, spherical or cylindrical. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013

Preparation of high molecular weight and elastic copolyester by reactive extrusion with diisocyanate compound for laser printing process

Abstract A branched copolyester was synthesized using dimethyl terephthalate (DMT), 2,2-bis[4-(2-hydroxypropoxy)phenyl]propane, ethylene glycol (EG) and 2-(hydroxymethyl)-2- ethylpropane-1,3-diol (trimethylol propane, TMP). The branched copolyester and p-phenylene diisocyanate (PPDI) were melt extruded to enhance the melt viscosity and elasticity for use as a toner binder in the laser printing process. The effects of PPDI content on melt, thermal and rheological properties of the chain-extended copolyester were investigated. The melt flow index (MI) decreased with increasing amount of PPDI, due to a reaction between the hydroxyl chain end and isocyanate group. The storage modulus, loss modulus and complex viscosity of the chain extended copolyester were higher and the modified Cole-Cole plots revealed the chain extended copolyester to have higher elasticity than that of the branched copolyester. The chain extended copolyester exhibited suitable melt and rheological properties for applications as a toner binder in the laser printing processes.

Poly[3-(3,4-dihydroxyphenyl)glyceric Acid] from Anchusa italica Roots

Elucidation of the main structural unit of a water-soluble, high-molecular weight preparation from the crude polysaccharides of Anchusa italica Retz. roots has been carried out. According to 13C NMR, 1H NMR and 2D heteronuclear 1H/13C HSQC spectral data, the main structural element of the high-molecular, water-soluble preparation was a regularly substituted polyoxyethylene chain, namely poly[oxy-1-carboxy-2-(3,4-dihydroxyphenyl)ethylene]. Most carboxylic groups of this caffeic acid-derived polymer of A. italica are methylated.

Low-molecular weight iron dextran and iron sucrose have similar comparative safety profiles in chronic kidney disease

Serious adverse events that occur with the administration of iron dextran are due to the high molecular weight preparations. Conclusions that iron sucrose and ferric gluconate are safer than iron dextran may be premature. Published literature comparing safety profiles of available parenteral iron products is reviewed. Administration of iron salts to pre-dialysis patients with chronic kidney disease may not be optimal. We recommend the total dose infusion of low molecular weight iron dextran as an option for iron replacement.

Preparation of high-molecular weight and high-sulfate content chitosans and their potential antioxidant activity in vitro

The influence of molecular weight and substitution degree of sulfated polysaccharides on their biological activity is considered in majority of works involving the anticoagulant or antiviral properties of these substances. Therefore, the present paper describes the effect of preparation conditions of sulfated chitosans on their molecular weight and sulfur content, such as different reaction time, acid solvent and temperature. Foregoing literature expounded the action of dichloroacetic acid (DCAA) as acid solvent in homogeneous reaction. However, DCAA is expensive and noxious, therefore, in the present paper cheap and non-noxious formic acid (88%) was in place of DCAA. Furthermore, during reaction formic acid was not dehydrated. Under formic acid we obtained the satisfying results that was higher yield and equivalent sulfur contents compared to DCAA. IR and 13C NMR spectrums proved the structure of the resultant obtained under formic acid or DCAA to be same. Now, it has not been reported for formic acid as acid solvent in homogeneous reaction of chitosan sulfatation. In this present paper, we also determined antioxidant activity of high-molecular weight and high-sulfate-content chitosans (HCTS). The results showed that HCTS could scavenge superoxide and hydroxyl radical. Its IC 50 is 0.012 and 3.269 mg/mL, respectively. It had obviously reducing power and slight chelating activity. The data obtained in in vitro models clearly establish the antioxidant potency of HCTS. It is a potential antioxidant in vitro.

Preparation of segmented, high molecular weight, aliphatic poly(ether-urea) copolymers in isopropanol. In-situ FTIR studies and polymer synthesis

The use of isopropanol (IPA) as the reaction solvent for the preparation of high molecular weight segmented polyether-urea copolymers based on cycloaliphatic diisocyanates was investigated. Reactivity of IPA with bis(4-isocyanatohexyl)methane (HMDI) and isophorone diisocyanate (IPDI) was studied between 0 and 40 °C using in-situ FTIR spectroscopy. HMDI, which has secondary isocyanate groups, shows a very slow reaction with a large excess of IPA at 0 and 23 °C. Analysis of the kinetic data indicates an activation energy of 51 kJ/mol for the reaction between HMDI and IPA. As expected, IPDI, which has both a primary and a secondary isocyanate (NCO) group, reacts faster with IPA compared with HMDI, which only has secondary NCO groups. However, the rate of reaction of IPDI with IPA at 0 °C is extremely slow (approximately 1% consumption of isocyanate in 60 min) thus allowing the use of IPA as the reaction solvent for polyether-urea synthesis. Preparation of high molecular weight, high-strength HMDI and IPDI based polyether-urea segmented copolymers in IPA has been demonstrated. Thermal analysis and stress–strain analyses were used to characterize the products.

Intra-articular hyaluronan: A disease modifying therapy?

Hyaluronan (HA) is a macromolecular glycosaminoglycan which is an important constituent of both synovial fluid and the extracellular matrix of cartilage. Research into the functions of HA have suggested a number of different biophysical, biochemical and cell regulatory roles in joint synovial tissues. Intra-articular HA therapy is now widely used in various countries for the treatment of osteoarthritis (OA) in both humans and animals. Numerous high purity pharmaceutical preparations are commercially available which have a variety of forms and molecular weights ranging from less than 1.0 × 106 Da (generally regarded as low molecular weight) up to 3–6.0 × 106 Da (high molecular weight). This range of molecular weights in the commercially available preparations is a direct reflection of the variation in results derived from both in vitro and in vivo studies of the use of HA in various experimental models. Intra-articular HA therapy has been registered by some companies as a device rather than a drug, which reflects both the initial theory that HA exerted its action through viscosupplementation of deficient synovial fluid, and the current scientific debate as to the specific therapeutic effects of HA on synovial tissues. Significant research is ongoing to further define the likely multiple actions of HA on joint tissues. The discovery that HA is rapidly cleared from joints after intra-articular injection, coupled with the realization that the symptomatic relief experienced by human OA patients treated by intra-articular HA, together suggest that viscosupplementation is not the only mode of action of HA. Studies in various animal models of inflammation and pain have supported an anti-inflammatory action of HA in joint disease. In vivo animal studies using models of OA suggest that low molecular weight HA provides better protection of cartilage than high molecular weight preparations. This is in direct contrast to the results of in vitro studies using chondrocyte or cartilage explant cultures in which high molecular weight HA preparations were found to be more effective at protecting cartilage from degradation than low molecular weight preparations. Evidence that low molecular weight forms of HA penetrate the joint tissues more deeply is a partial explanation for this, as it is likely that the low molecular weight HA is able to achieve higher concentrations of the drug in the pericellular environment. However, recent research has provided additional evidence of specific interaction between HA, various cell receptors such as CD44. This, together with other studies in cell biology which have demonstrated that signal transduction and gene expression arise from HA receptor binding suggest an immunoregulatory role for HA. Our present knowledge of interactions of HA at the cellular and molecular level is still inadequate, but there is strong evidence from animal model experiments as well as from studies on human synovial biopsies that the positive effects exhibited by HA in OA joints are largely mediated by its anti-inflammatory, cytoprotective and immunoregulatory properties. There is significant scientific evidence that intra-articular HA therapy can provide cartilage protection. However, this probably does not arise from a direct effect of HA on chondrocytes, but rather is secondary to the down regulation of cytokine, free radical and proteolytic activities in the synovial fluid and synovium, all of which can adversely affect chondrocyte metabolism.

Fighting Infectious Diseases with Inhibitors of Microbial Adhesion to Host Tissues

The majority of infectious diseases are initiated by the adhesion of pathogenic organisms to the tissues of the host. In many cases this adhesion is mediated by lectins present on the surface of the infectious organism that bind to complementary carbohydrates on the surface of the host tissues. Soluble carbohydrates recognized by the bacterial lectins block the adhesion of the bacteria to animal cells in vitro. Moreover, such carbohydrates have been shown to protect against experimental infection by lectin–carrying bacteria of different mammals, such as mice, rabbits, calves, and monkeys. Agents other than carbohydrates also block adhesion, as demonstrated with cranberry juice as well as with low and high molecular weight preparations isolated from the juice. Both kinds of preparation inhibited the adhesion in vitro of Escherichia coli to different animal cells. In addition, the high molecular weight material acted similarly on the adhesion of Helicobacter pylori to human gasrointenstinal cells, and on the coaggregation of oral bacteria. Furthermore, in limited clinical studies regular drinking of cranberry juice had a significant preventive effect on bacteriuria, and the high molecular weight constituent of the juices was also effective in decreasing the salivary level of Streptococus mutans, the major cause of tooth decay. Because the inhibitors of adhesion examined are not bactericidal, the selection of resistant inhibitor strains is unlikely to occur. Together, these findings may lead to new therapeutic strategies that are in dire need because of the world–wide increase in antibiotic resistant bacteria.

A routine high-performance size-exclusion chromatography to determine molecular size distribution of Haemophilus influenzae type b conjugate vaccines

High-performance size exclusion chromatography has been used to determine the molecular size distribution of Haemophilus influenzae type b (Hib) conjugate vaccines. Both high molecular weight preparations of native Hib capsular polysaccharide coupled to tetanus toxoid and low molecular weight vaccines with Hib oligosaccharides linked to the CRM 197 nontoxic mutant diphtheria protein were analysed. Columns with different fractionation ranges were used for the two kinds of vaccines. This method showed to be rapid, accurate and reproducible for different lots of Hib vaccine of different composition produced by various manufacturers. It could replace more time-consuming chromatographic methods enabling control authorities to employ a single methodological approach for different Hib vaccines.

Preparation and characterization of water soluble high molecular weight β-cyclodextrin-epichlorohydrin polymers

Water soluble β-cyclodextrin polymers were prepared from β-cyclodextrin crosslinked with epichlorohydrin under basic conditions. The influence of preparation conditions on the molecular weight distribution has been investigated. The knowledge of this relationship allows for the choice of reaction parameters according to the desired weight distribution of the β-cyclodextrin polymer. A procedure was developed for the preparation of high molecular weight water soluble polymer ( Mw higher than 10 4).

Preparation of megabase‐size DNA from plant nuclei

A novel technique has been developed for the preparation of high molecular weight (HMW) DNA from plant nuclei. This technique involves physical homogenization of plant tissues, nuclei isolation, embedding of the nuclei in low‐melting‐point agarose microbeads or plugs, and DNA purification in situ. This technique is simple, rapid, and economical, and the majority of the DNA prepared is over 5.7 Mb in size. The genomic DNA content of the HMW DNA prepared by this technique is enriched by at least threefold and the chloroplast DNA content is reduced by over twofold relative to that prepared from plant protoplasts by existing methods. The DNA is readily digestible with different restriction enzymes and partial digestions of the DNA could be reproducibly performed. This method has been successfully used for the preparation of HMW DNA from a wide range of plant taxa, including grasses, legumes, vegetables, and trees. These results demonstrate that the DNA prepared by this technique is suitable for plant genome analysis by pulsed‐field gel electrophoresis and for the construction of yeast and bacterial artificial chromosomes.

Polymorphism of Nuclear DNA from Various Rice Cultivars, as Detected by Staining of Gels after Pulsed-field Gel Electrophoresis.

A new method was developed for preparation of highmolecularweight (HMW) DNA from rice using young or mature leaves as source material. Compared with the ori-ginal method, in which germ' tissues were used as source material (Hatano et al., 1992), the new method is simpler and faster because the procedure for isolation of germ tissues is no longer necessary. Moreover, even if a limited supply of seeds is expected, HMW DNA can be prepared from mature leaves derived from a smaller number of seeds. Therefore, the method is suitable for screening genomes of a number of varieties of rice for analysis by pulsedfield gel electrophoresis. HMW DNA from rice varieties that included nine Japonica cultivars, one Indica cultivar, and one strain of Oryza puuctata, a wild rice was prepared by the newly developed method. Each of the samples was digested with restriction enzymes and fragments were separated by pulsedfield gel electrophoresis. After staining of gels, a microfine banding pattern was visualized over a smeared background in each lane. Each pattern was specific for a specific variety of rice. Furthermore, although similarities were found among the Japonica cultivars tested, few common microbands were observed among the Indica cultivar, the strain of Oryza punctata and the group of Japonica cultivars tested. These observa-tions indicate that a high degree of polymorphism of nuc-lear DNA among rice varieties can be detected by simple staining of gels after pulsed-field gel electrophoresis.

Preparation of high molecular weight radioiodinated alginic acid

AbstractA simple procedure is described for preparing125I‐labelled alginic acid. The reducing terminal of alginic acid is derivatized with tyramine through the formation of Schiff base which is subsequently reduced with sodium cyanoborohydride. By radioiodination of the aromatic ring introduced,125I‐labelled alginic acid was obtained with high specific activity.

Preparation and Characterization of the High Molecular Weight [3H]Hyaluronic Acid

Two methods for the preparation of high molecular weight [3H]hyaluronic acid were investigated. In the first one, hydrogen atoms in the molecule were replaced by tritium. This isotopic substitution was performed in aqueous solution using Pd/CaCO3 as the catalyst. In the second method, the high molecular weight hyaluronic acid was alkylated with [3H]methyl bromide in liquid ammonia at a temperature of -33.5 °C. High-performance gel permeation chromatographic separation method was used for the isolation and characterization of the high molecular weight [3H]hyaluronic acid. Molecular weight parameters for the labelled biopolymers were Mw = 128 kDa, Mw/Mn = 1.88 (first method) and Mw = 268 kDa, Mw/Mn = 1.55 (second method). The high molecular weight [3H]hyaluronic acid having Mw = 268 kDa was degraded further by specific hyaluronidase. Products of the enzymatic depolymerization were observed to be identical for both, labelled and cold biopolymer. This finding indicates that the described labelling procedure using [3H]methyl bromide does not induce any major structural rearrangements in the molecule.

Pregnancy-Associated Plasma Protein-A-Induced Inhibition of Human Leukocyte Elastase: An Artifact

Pregnancy-associated plasma protein A (PAPP-A), was reported to be an inhibitor in many in vitro systems. Since it was shown that the inhibition of coagulation and complement activity attributed to PAPP-A was in fact due to a contamination by heparin occurring during the purification process, we undertook the present study to see whether the reported PAPP-A-induced inhibition of human leukocyte elastase (HLE) could also be attributed to heparin contamination. PAPP-A was purified from maternal pregnancy EDTA plasma by a method which was previously shown to eliminate contaminating heparin: this preparation was inactive in the HLE assay. But PAPP-A isolated by heparin-Sepharose chromatography, or a PAPP-A-free washing of the heparin-Sepharose column were both inhibitors of HLE. Furthermore the inactive PAPP-A preparation, when incubated with the PAPP-A-free washing of the heparin-Sepharose column, yielded a high molecular weight preparation which inhibited HLE. It is concluded that PAPP-A is not an inhibitor of HLE and that the inhibition of HLE previously attributed to PAPP-A was due to contaminating heparin.

Facile preparation of high molecular weight, highly conductive poly(2,5-furylene vinylene)

High molecular weight, freestanding films of poly(2,5-furylene vinylene)(PFV) are readily prepared via a water soluble precursor polymer, are electrochemically reversibly donor and acceptor dopable, and reach conductivities as high as 36 S/cm on acceptor doping.