Molecular Size Profile Research Articles

Hunting for the signatures of cancer by plasma DNA sequencing

Liquid biopsy refers to the analysis of DNA released by tumours into the circulation of patients for the detection, prognostication, selection of therapeutic targets or monitoring of malignancies. For example, regulatory approval has been granted for the detection of epidermal growth factor receptor mutations in plasma of patients with non-small cell lung cancers as a means to stratify therapy. However, the key challenge in the diagnostics of cancer is about its early detection. Given that cancers are highly heterogeneous, our group has been developing whole genome approaches to detect the signatures of cancer via a liquid biopsy. The rationale is to extract as much of the molecular information as possible from a plasma sample to catch a glimpse of the possible presence of cancer. Genome-wide approaches to detect tumour-associated copy number aberrations, molecular size profile, DNA methylation signatures and transcriptomic features from circulating nucleic acids have been developed. We have further developed an approach to pinpoint the anatomical location of malignancy, naming to determine a tissue map non-invasively. In summary, many facets of molecular information could be extracted from a liquid biopsy.Acknowledgement: Supported by the Research Grants Council of the Hong Kong SAR Government under the Theme-based research scheme (T12-404/11). Liquid biopsy refers to the analysis of DNA released by tumours into the circulation of patients for the detection, prognostication, selection of therapeutic targets or monitoring of malignancies. For example, regulatory approval has been granted for the detection of epidermal growth factor receptor mutations in plasma of patients with non-small cell lung cancers as a means to stratify therapy. However, the key challenge in the diagnostics of cancer is about its early detection. Given that cancers are highly heterogeneous, our group has been developing whole genome approaches to detect the signatures of cancer via a liquid biopsy. The rationale is to extract as much of the molecular information as possible from a plasma sample to catch a glimpse of the possible presence of cancer. Genome-wide approaches to detect tumour-associated copy number aberrations, molecular size profile, DNA methylation signatures and transcriptomic features from circulating nucleic acids have been developed. We have further developed an approach to pinpoint the anatomical location of malignancy, naming to determine a tissue map non-invasively. In summary, many facets of molecular information could be extracted from a liquid biopsy. Acknowledgement: Supported by the Research Grants Council of the Hong Kong SAR Government under the Theme-based research scheme (T12-404/11).

A tale of locust swarms, cannibals, ageing and human obesity

Liquid biopsy refers to the analysis of DNA released by tumours into the circulation of patients for the detection, prognostication, selection of therapeutic targets or monitoring of malignancies. For example, regulatory approval has been granted for the detection of epidermal growth factor receptor mutations in plasma of patients with non-small cell lung cancers as a means to stratify therapy. However, the key challenge in the diagnostics of cancer is about its early detection. Given that cancers are highly heterogeneous, our group has been developing whole genome approaches to detect the signatures of cancer via a liquid biopsy. The rationale is to extract as much of the molecular information as possible from a plasma sample to catch a glimpse of the possible presence of cancer. Genomewide approaches to detect tumour-associated copy number aberrations, molecular size profile, DNA methylation signatures and transcriptomic features from circulating nucleic acids have been developed. We have further developed an approach to pinpoint the anatomical location of malignancy, naming to determine a tissue map non-invasively. In summary, many facets of molecular information could be extracted from a liquid biopsy.

Molecular study of Trypanosoma caninum isolates based on different genetic markers.

Trypanosoma caninum is a parasite recently described in dogs, whose life cycle is rather unknown. Here, we performed a genetic study with T. caninum samples obtained in different Brazilian regions. The study was based on PCR assays target to small and large subunit ribosomal DNA (rDNA) (18S rDNA and 24Sα rDNA), cytochrome B (Cyt b), and internal transcribed spacer 1 rDNA (ITS1 rDNA) following by the sequence analysis. Additionally, we used primers for the variable regions of kinetoplast DNA (kDNA) minicircles and endonucleases restriction in the ITS1 rDNA amplification product. T. caninum samples displayed the same patterns. Tree construction confirmed the close relationship between T. caninum samples, regardless of the molecular target used and endonuclease restriction digestion revealed that all samples have the same restriction profile. Therefore, T. caninum seems to be a genetically homogeneous specie. In the kDNA assay, T. caninum possessed a different molecular size profile with respect to others trypanosomes, 330 and 350 bp. This study provides nucleotide sequences from different regions of the genome of T. caninum that certainly facilitate future studies.

Hyaluronan Metabolism in Human Keratinocytes and Atopic Dermatitis Skin Is Driven by a Balance of Hyaluronan Synthases 1 and 3

Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.

Phylogenetic Analysis of Bolivian Bat Trypanosomes of the Subgenus Schizotrypanum Based on Cytochrome b Sequence and Minicircle Analyses

The aim of this study was to establish the phylogenetic relationships of trypanosomes present in blood samples of Bolivian Carollia bats. Eighteen cloned stocks were isolated from 115 bats belonging to Carollia perspicillata (Phyllostomidae) from three Amazonian areas of the Chapare Province of Bolivia and studied by xenodiagnosis using the vectors Rhodnius robustus and Triatoma infestans (Trypanosoma cruzi marenkellei) or haemoculture (Trypanosoma dionisii). The PCR DNA amplified was analyzed by nucleotide sequences of maxicircles encoding cytochrome b and by means of the molecular size of hyper variable regions of minicircles. Ten samples were classified as Trypanosoma cruzi marinkellei and 8 samples as Trypanosoma dionisii. The two species have a different molecular size profile with respect to the amplified regions of minicircles and also with respect to Trypanosoma cruzi and Trypanosoma rangeli used for comparative purpose. We conclude the presence of two species of bat trypanosomes in these samples, which can clearly be identified by the methods used in this study. The presence of these trypanosomes in Amazonian bats is discussed.

Acid hydrolysis of commercial chitosans

AbstractCommercial chitosans were subjected to controlled acid hydrolysis and their degrees of deacetylation (DD), molecular size and rheological flow profiles determined (pre‐ and post‐hydrolysis) by 1H‐NMR spectroscopy, high‐performance size‐exclusion chromatography and rheometry, respectively. Hydrolysis resulted in DD increases between 4 and 11%. Unhydrolysed chitosans had Mw and Mn values in the ranges 700–1200 and 130–210 kDa, respectively. Chitosan with the smallest initial molecular size averages had the smallest averages after hydrolysis; however, a chitosan with an intermediate initial molecular size proved to be most resistant to hydrolysis. Molecular size trends were paralleled by zero shear viscosity (η0) measurements determined by application of the Williamson model to rheological flow profile data. Viscosity is obviously related to molecular size, but does not necessarily reflect relative ease of hydrolysis, since specific hydrolysis conditions affect structurally similar polysaccharides in different ways (in terms of rate of depolymerisation and de‐N‐acetylation, etc), which are not simply due to differences in molecular size profiles pre‐hydrolysis. Copyright © 2005 Society of Chemical Industry

Alginate fibres modified with unhydrolysed and hydrolysed chitosans for wound dressings

A range of commercial chitosans were sourced, subjected to controlled acid hydrolysis, and their molecular size profiles and degrees of acetylation (DA) determined (pre- and post-hydrolysis) by High Performance Size Exclusion Chromatography and 1H-NMR spectroscopy, respectively. Unhydrolysed and hydrolysed chitosans were subsequently utilised for modification of sodium alginate/alginic acid fibres (prepared using a range of different fibre spinning conditions), and levels of chitosan incorporated onto/into base alginate fibres were estimated by elemental analysis. Tensile properties (% elongation and tenacity) of resultant chitosan/alginate fibres were determined in order to assess their suitability for potential application in wound dressings. A broad range of chitosan contents (∼0–6% w/w) and hydrolysed chitosan contents (∼7–25% w/w) were obtained using a variety of alginate and chitosan starting materials. Modification of fibres with unhydrolysed chitosans generally resulted in a significant reduction in tenacity (and a reduction in % elongation if a water washing stage was not used), i.e. no increase in fibre strength was observed, implying that the unhydrolysed chitosan is more like a coating rather than penetrating/reinforcing the alginate fibre. Reduction of chitosan molecular weight had a positive effect on its ability to penetrate the alginate fibres, not only increasing fibre chitosan content, but also reinforcing fibre structure and thus enhancing tensile properties (compared with unhydrolysed chitosan/alginate fibres). Hydrolysed chitosan/alginate fibres demonstrated an antibacterial effect (in terms of bacterial reduction) with initial use, and had the ability to provide a slow release/leaching of antibacterially active components (presumably hydrolysed chitosan fragments). The overall aims of maximising chitosan content (in order to provide satisfactory antibacterial activity) whilst retaining desirable physical properties (i.e. satisfactory textile processing ability) were therefore achieved, with respect to the fibres having potential application as wound dressing materials.

Vase solutions containing sucrose result in changes to cell walls of sandersonia (Sandersonia aurantiaca) flowers

Abstract It is well established that vase solutions containing sugar can improve the vase-life of many cut flower crops. Since cut sandersonia flowers supplied with 2% sucrose are firmer during wilting compared to water-fed controls, we have examined whether the effects of sucrose treatment extend to alterations in cell wall structure in the floral tissues, which may influence the wilting-related flower softening. Mature but not fully opened individual flowers were removed from the stems of sandersonia plants and were fed continuously with either 2% sucrose solution or water for up to 10 days. Sucrose supplementation resulted in decreased amounts of chelator-soluble pectin and increased amounts of Na2CO3-soluble pectin per individual flower, and also changed the molecular size profiles of both these pectin fractions compared to the water-fed controls. The molecular size differences were obvious after 3 days in vase solutions, and diminished with subsequent vase time. Senescence-related galactose loss was delayed in sucrose-fed flowers but there was no difference in the levels of β-galactosidase activity present in these flowers compared to controls. The observed differences in cell wall pectins due to sucrose feeding were not reflected in differences to the overall firmness of pre-senescent flowers (up to day 3). High levels of galactose persisted into the wilting phase when sucrose-fed flowers were firmer than water-fed controls. We conclude that while sucrose induced significant quantitative and qualitative differences in pectin fractions and galactose content, firmness of floral tissue, particularly during senescence, was not governed by these events alone.

MALDI-MS Characterization of Maltooligo/polysaccharides From Debranched Starch Amylopectin of Corn and Barley

Aprotocol was developed for molecular characterization of debranched starch amylopectin using Matrix-assisted laser desorption/ionizationmass spectrometry (MALDI-MS). Starch from waxy corn was used in the protocol development. Various factors influencing the protocol were investigated and discussed. Using the developed protocols, starch amylopectin from barley grains (waxy-type Candle and regular-type Phoenix) were also characterized. The study suggested that MALDI-MS is suitable to determine the molecular size profile of debranched starch (maltooligo- and polysaccharides) faster and with greater accuracy than any present analytical methodology.

Cell wall modifications, degrading enzymes and softening of carambola fruit during ripening

The fruit extracts of the slow-ripening cv. B 10 carambola (Averrhoa carambola L.) contained a number of cell wall hydrolases. The predominant ones appeared to be β-(1,4)-glucanase (as carboxymethylcellulase), pectinesterase, β-galactosidase, and polygalacturonase (PG). Other significant hydrolases the activity of which also increased with ripening were the glycosidases, α-arabinosidase, α-galactosidase, and α-mannosidase, and also the glycanases, β-(1,4)-galactanase and xylanase. Throughout ripening, as pectins and hemicelluloses were being differentially modified, the levels of buffered-phenol cell wall materials, total polyuronides as well as arabinose, galactose, xylose, and glucose decreased. At early ripening phase (days 0–12) there was no apparent pectin solubilization, and the loosely-bound water- and chelator-soluble pectins were the first pectic polysaccharides to be affected. That of the former exhibited an upshift, whilst the latter, showed a distinct downshift in their molecular size profiles. At late ripening phase (days 12–24) when tissue firmness had declined substantially, dramatic changes involving pectins and hemicelluloses were evident. Pectins were solubilized, and this increased solubility was accompanied by depolymerization of all pectin classes and a decrease in the level of the Na2CO3-soluble polyuronides. Coincident with this marked modifications of the tightly-bound, predominant polyuronide fractions and hemicellulose was the increase in activities of polygalacturonase and β-(1,4)-glucanase, suggesting that these enzymes may contribute to wall modifications late during ripening. Some of the other wall hydrolases, namely, α-arabinosidase, α-galactosidase and certain isoforms of β-galactosidase/galactanase, might be relevant to the early ripening changes when pectin solubilization was limited.

EFFECT OF PHOTOOXIDATION ON MOLECULAR WEIGHT DISTRIBUTIONS OF CORBETT ASPHALT FRACTIONS BY GEL PERMEATION CHROMATOGRAPHY

ABSTRACT Oxidation of asphalt is believed to be one of the major contributing factors to the failure of asphalt pavement. Due to molecular complexity, the oxidation mechanism of asphalt is complicated by interactions among the molecules of different types. To minimize the interactions, the asphalt sample was separated into saturates, naphthalene aromatics, polar aromatics, and asphaltenes. These Corbett fractions were then oxidized under ultraviolet irradiation. Gel permeation chromatography was used to analyze the molecular weight changes of the Corbett fractions during photooxidation. All Corbett fractions have shown significant increase in molecular weight. The oxidized saturates have the highest weight average molecular weight and the most complicated molecular size profiles among the oxidized Corbett fractions

COMPARISON OF MOLECULAR WEIGHT DISTRIBUTIONS OF CORBETT ASPHALT FRACTIONS BY GEL PERMEATION CHROMATOGRAPHY

Accurate molecular weight distributions are essential to the determination of the molecular structures of asphalt. Due to molecular complexity and interaction among the molecules of asphalt, precise measurement for the molecular weights of asphalt can not be achieved. In order to minimize the interactions of asphaltic materials, a petroleum asphalt sample was separated into saturates, naphthalene aromatics, polar aromatics, and asphaltenes. These Corbett fractions were then analyzed by gel permeation chromatography against nondispersive polystyrene standards. The molecular weights and the molecular size profiles were obtained. Among these Corbett fractions, naphthalene aromatics have the lowest number average molecular weight; saturates have the lowest weight average molecular weight and dispersity; and asphaltenes have the highest number and weight average molecular weights and dispersity.

A simple method for affinity purification of radiolabeled monoclonal antibodies

A simple method is described for affinity purification of radiolabeled antibodies using glutaraldehyde-fixed tumor target cells. The cell-bound antibody fraction is removed from the cells by an acid wash and then immediately subjected to buffer-exchange chromatography. The method was applied to the D3 murine monoclonal antibody which binds to a 290 kDa antigen on the surface of Line 10 guinea pig carcinoma cells. No alteration in the molecular size profile was detected after acid washing. Purification resulted in a significant increase in immunoreactivity by an average of 14 ± 47% (SD; range 4–30%).

Fructans in Forage Grasses with Varying Degrees of Coldhardiness

Summary Changes in WSC content and molecular size profile of fructan in perennial and annual forage grasses ofvarying coldhardiness were determined during the fall months of 1987. Phleum, Phalaris and Bromus spp. are hardier than Dactylis, Festuca and Lolium spp. The concentration of WSC in herbage on August 5 was much higher in annual ryegrass than perennials. There was no linear relationship between the WSC content in basal 5 cm top tissues and the coldhardiness. With TLC, a series of low-DP fructans were detected in basal top tissues of Bromus, Festuca and Lolium spp., but only very small amounts occurred in Phleum, Phalaris and Dactylis . There was little change in low DP fructans (DP 3 to 10) during the fall season. HPLC showed four distinct types of molecular size distribution pattern of fructans: Phleum-Phalaris type, Dactylis type, Bromus type and Festuca-Lolium type. A typical change in HPLC profile in hardy grasses during the fall months was a decrease in high-DP fructan (DP ≥ 35) and an increase in medium-DP fructan (DP 11 to 34).

Biochemical changes in periodontal ligament ground substance associated with short-term intrusive loadings in adult monkeys ( Macaca fascicularis)

Proteoglycan-like fractions (PG) were isolated from the ligaments of teeth undergoing various degrees of intrusive loadings. The PG were characterized by their molecular-size profiles on Sepharose 4B, the presence of uronic acid in the separated fractions and by the electrophoretic detection of constituent glycosaminoglycans including heparan sulphate, hyaluronic acid, dermatan sulphate and chondroitin-4-sulphate. The high molecular-weight fraction, peak i (estimated minimum size, 2 × 10 6 daltons) of the normal-functioning (stressed) ligament was reduced approx. 70 per cent, compared with ligament undisturbed for 3 h. There was a decrease in peak-i size between 0.25 and 1 N loadings of approx. 72 per cent. The 4 N loadings produced a further decrease followed by an increase during a 3 h undisturbed recovery phase. Thus changes in the chemistry and properties of the ground-substance components of the periodontal ligament could partly explain changes in tooth mobility.

Serologic aspects of IgG4 antibodies. II. IgG4 antibodies form small, nonprecipitating immune complexes due to functional monovalency.

Human IgG4 antibodies directed against phospholipase A, the P1 antigen from Dermatophagoïdes pteronyssinus extracts, and cat albumin were found unable to cross-link antigen. Previously, it was demonstrated that IgG4 antibodies, in contrast to IgG1 antibodies, did not cross-link Sepharose-bound antigen and antigen added in solution. To eliminate the possibility that this phenomenon was caused by preferential binding of both IgG4 Fab fragments to the solid-phase-bound antigen, cross-linking of antigen was studied in a fluid-phase system. In this test, incapability of IgG4 antibodies to bridge two antigens was also found. As a result of such a phenomenon, it is expected that immune complexes formed by IgG4 antibodies will be considerably smaller than complexes formed by IgG1. This was confirmed by analysis of the molecular size profiles of IgG1- and IgG4-containing immune complexes in sucrose-density gradients. Moreover, IgG1 was able to precipitate antigen in a radioimmunoprecipitation test, whereas precipitation was not demonstrable by the same amount of IgG4 antibodies. Even 3% polyethylene glycol 8,000 did not precipitate the small IgG4-containing immune complexes efficiently. The antibodies studied were of a high-affinity type, and there was no significant difference in association constants between IgG1 and IgG4 antibodies. Therefore, we were not able to confirm observations reported in the literature that the IgG4 subclass is associated with a low-affinity antibody response; probably, the affinity of the IgG4 antibodies was underestimated by other investigators because of the polyethylene glycol precipitation technique used to separate antibody-bound and free antigen. Our findings stress the point that IgG4 antibodies take a special place in the immune response upon chronic exposure to antigen.

The effect of steroids on the size heterogeneity of pituitary prolactin in castrate male rats

Male Sprague-Dawley rats were castrated and given daily sc. injections of estradiol (E 2, 10 μg/day), testosterone propionate (TP, 1.0 mg/day), dihydrotestosterone (DHT, 1.0 mg/day) or sesame oil (SO, 0.2 ml/day). A group of sham castrate males received daily sc. injections of SO (0.2 ml/day). On day 8 of steroid treatment animals were decapitated and anterior pituitaries were removed and hemisected. Each half was homogenized in PBS buffer (0.01 M Na 2HPO 4-NaH 2PO 4; 0.14 M NaCl; 0.1% bovine serum albumin) at either pH 7.6 or 10.6. Homogenates were chromatographed on Sephadex G-100 columns and eluted fractions were assayed for prolactin (PRL) by RIA. Four immunoreactive forms of PRL, designated as “void volume,” “big big,” “big” and “little,” were eluted from the pituitary homogenates of each experimental group. Homogenates obtained at pH 7.6 contained a greater percentage of PRL in the “void volume” and less activity in the “big” and “little” forms than pH 10.6 homogenates in all experimental groups. Pituitaries from SO- and TP-treated castrate animals contained significantly greater percentages of activity in the “void volume” at pH 7.6 compared to the other groups. At pH 10.6, the pituitary homogenates from the E 2-treated group eluted a significantly greater percentage of “big” PRL and a smaller percentage of “little” PRL compared to all other groups. These findings suggest that androgenic and estrogenic steroids may play a role in the pituitary PRL molecular size profile of the male rat.

Association—dissociation problems in characterizing asphaltenes in coal liquids

The determination of asphaltenes in coal liquefaction products is strongly related to the chosen solvents, extraction time, sequence of extractive solvents, and co-solvent effects of the coal liquids. Recognizing that asphaltenes are operationally defined, the above observations raise the question of whether there are “asphaltenes” in the unseparated coal liquids or whether they are artifacts of the separation procedure, resulting from the association of unstable, reactive molecules that combine upon their concentration and isolation from the original slurry. Using preparative and analytical Gel Permeation Chromatography and solvent extraction procedures, qualitative molecular size profiles of coal liquid products have been obtained. It has been concluded that: (1) the pre-asphaltenes, asphaltenes and oils exist in the coal liquids, and are not artifacts of the separation procedures; (2) their molecular sizes follow the order pre-asphaltenes > asphaltenes > oils; (3) association—dissociation of the coal liquids is indicated and (4) coal liquid fractions obtained by different methods should not be considered equivalent.