Low molecular weight heparins are complex polycomponent drugs that have recently become amenable to top-down analysis using liquid chromatography-mass spectrometry. Even using open source deconvolution software, DeconTools, and automatic structural assignment software, GlycReSoft, the comparison of two or more low molecular weight heparins is extremely time-consuming, taking about a week for an expert analyst and provides no guarantee of accuracy. Efficient data processing tools are required to improve analysis. This study uses the programming language of Microsoft Excel™
Visual Basic for Applications to extend its standard functionality for macro functions and specific mathematical modules for mass spectrometric data processing. The program developed enables the comparison of top-down analytical glycomics data on two or more low molecular weight heparins. The current study describes a new program, GlycCompSoft, which has a low error rate with good time efficiency in the automatic processing of large data sets. The experimental results based on three lots of Lovenox®, Clexane® and three generic enoxaparin samples show that the run time of GlycCompSoft decreases from 11 to 2 seconds when the data processed decreases from 18000 to 1500 rows.
Hyperkalemia is a potentially life-threatening condition, and patients who have chronic kidney disease, who are diabetic, or who are taking renin–angiotensin–aldosterone system inhibitors are at increased risk. Therapeutic options for hyperkalemia tend to have limited effectiveness and can be associated with serious side effects. Colonic potassium secretion can increase to compensate when urinary potassium excretion decreases in patients with renal impairment, but this adaptation is insufficient and hyperkalemia still results. Patiromer is a novel, spherical, nonabsorbed polymer designed to bind and remove potassium, primarily in the colon, thereby decreasing serum potassium in patients with hyperkalemia. Patiromer has been found to decrease serum potassium in patients with hyperkalemia having chronic kidney disease who were on renin–angiotensin–aldosterone system inhibitors. Results of nonclinical studies and an early phase clinical study are reported here. Two studies with radiolabeled drug, one in rats and the other in dogs, confirmed that patiromer was not absorbed into the systemic circulation. Results of an in vitro study showed that patiromer was able to bind 8.5 to 8.8 mEq of potassium per gram of polymer at a pH similar to that found in the colon and had a much higher potassium-binding capacity compared with other resins, including polystyrene sulfonate. In a study in hyperkalemic rats, a decrease in serum potassium was observed via an increase in fecal potassium excretion. In a clinical study in healthy adult volunteers, a significant increase in fecal potassium excretion and a significant decrease in urinary potassium excretion were observed. Overall, patiromer is a high-capacity potassium binder, and the chemical and physical characteristics of patiromer may lead to good clinical efficacy, tolerability, and patient acceptance.
patiromer; hyperkalemia; chronic kidney disease; RAASi; potassium
Systemic sclerosis (SSc) caused fibrosis can be fatal and it still lack of effective treatment. Hydrogen sulfide (H2S) appears to be an attractive therapeutic candidates. This study aimed to investigate the protective effect of H2S on SSc-associated skin and lung fibrosis.
We developed a model of SSc by subcutaneous injecting BLM to female C3H mice. The mice received daily subcutaneous injections of NaHS (56 and 112 μg/kg), an H2S donor. On days 7, 28, and 42, the mice were killed and blood samples were collected to measure the plasma H2S concentration, the skin and lung tissues was harvested for microscopic examination, immunohistochemistry and quantify biological parameters (hydroxyproline content, RT-qPCR and Western blot).
In model group, the dermis of skin tissues at different time points gradually thickened, collagen deposition increased. The lung tissues presented pathological changes such as obvious inflammatory cell infiltration, increased collagen deposition and the plasma H2S concentrations points significantly decreased. Administration of NaHS markedly decreased the biomarkers of fibrosis such as α-smooth muscle actin, collagen-I, collagen-III, fibronectin, transforming growth factor-β1, Smad2/3 phosphorylation and inflammation including the marker protein of monocyte/macrophage and monocyte chemoattractant protein-1 in the lung. Compared to the low dose group, the expression in the high dose group have decreased trend, but the difference was not significant.
We demonstrate the beneficial effects of H2S on SSc-associated skin and lung fibrosis. H2S may be a potential therapy against this intractable disease.
Systemic sclerosis; Fibrosis; Transforming growth factor-β1; Hydrogen sulfide
Heparin is the most widely used anticoagulant drug in the world today. Heparin is currently produced from animal tissues, primarily porcine intestines. A recent contamination crisis motivated development of a non-animal-derived source of this critical drug. We hypothesized that Chinese hamster ovary (CHO) cells could be metabolically engineered to produce a bioengineered heparin, equivalent to current pharmaceutical heparin. We previously engineered CHO-S® cells to overexpress two exogenous enzymes from the heparin/heparan sulfate biosynthetic pathway, increasing the anticoagulant activity ~100-fold and the heparin/heparan sulfate yield ~10-fold. Here, we explored the effects of bioprocess parameters on the yield and anticoagulant activity of the bioengineered GAGs. Fed-batch shaker-flask studies using a proprietary, chemically-defined feed, resulted in ~two-fold increase in integrated viable cell density and 70% increase in specific productivity, resulting in nearly three-fold increase in product titer. Transferring the process to a stirred-tank bioreactor increased the productivity further, yielding a final product concentration of ~90 µg/mL. Unfortunately, the product composition still differs from pharmaceutical heparin, suggesting that additional metabolic engineering will be required. However, these studies clearly demonstrate bioprocess optimization, in parallel with metabolic engineering refinements, will play a substantial role in developing a bioengineered heparin to replace the current animal-derived drug.
CHO cells; Disaccharide analysis; Fed-batch cultures; Glycosaminoglycans; Metabolic engineering
Pneumococcal type-3 polysaccharide (Pn3P) is considered a major target for the development of a human vaccine to protect against Streptococcus pneumonia infection. Thus, it is critical to develop methods for the preparation and analysis of Pn3P-derived oligosaccharides to better understand its immunological properties. In this paper, we profile oligosaccharides, generated by the free radical depolymerization of Pn3P, using liquid chromatography (LC)-tandem mass spectrometry (MS/MS). Hydrophilic liquid interaction chromatography (HILIC)-mass spectrometry (MS) revealed a series of oligosaccharides with an even- and odd-number of saccharide residues, ranging from monosaccharide, degree of polymerization (dp1) to large oligosaccharides up to dp 20, generated by free radical depolymerization. Isomers of oligosaccharides with an even number of sugar residues were easily separated on a HILIC column, and their sequences could be distinguished by comparing MS/MS of these oligosaccharides and their reduced alditols. Fluorescent labeling with 2-aminoacridone (AMAC) followed by reversed phase (RP)-LC-MS/MS was applied to analyze and sequence poorly separated product mixtures, as RP-LC affords higher resolution of AMAC-labeled oligosaccharides than does HILIC-based separation. The present methodology can be potentially applied to profiling other capsular polysaccharides.
Pneumococcal type 3 polysaccharide; free radical depolymerization; fluorescent labeling; liquid chromatography-mass spectrometry; isomeric oligosaccharides
The B3LYP/6-311+G(d)-SDD method, which considers the relativistic effect of bromine, was adopted for the calculations of the selected polybrominated diphenyl ethers (PBDEs) in the present study, in which the B3LYP/6-311+G(d) method was also applied. The calculated values and experimental data for structural parameters of the selected PBDEs were compared to find the suitable theoretical methods for their structural optimization. The results show that the B3LYP/6-311+G(d) method can give the better results (with the root mean square errors (RMSEs) of 0.0268 for the C–Br bond and 0.0161 for the C–O bond) than the B3LYP/6-311+G(d)-SDD method. Then, the B3LYP/6-311+G(d) method was applied to predict the structures for the other selected PBDEs (both neutral and anionic species). The lowest unoccupied molecular orbital (LUMO) and the electron affinity are of a close relationship. The electron affinities (vertical electron affinity and adiabatic electron affinity) were discussed to study their electron capture abilities. To better estimate the conversion of configuration for PBDEs, the configuration transition states for BDE-5, BDE-22 and BDE-47 were calculated at the B3LYP/ 6-311+G(d) level in both gas phase and solution. The possible debromination pathway for BDE-22 were also studied, which have bromine substituents on two phenyl rings and the bromine on meta-position prefers to depart from the phenyl ring. The reaction profile of the electron-induced reductive debromination for BDE-22 were also shown in order to study its degradation mechanism.
relativistic effects; debromination; pseudo-potentials; transition state; adiabatic electron affinity; vertical electron affinity
The determination of complex analytes, present at low concentrations, in biological fluids poses a diffcult challenge. This study relies on an optimized method of recovery, enzymatic treatment, and disaccharide analysis by liquid chromatography–tandem mass spectrometry to rapidly determine low concentrations of glycosaminoglycans in human urine. The approach utilizes multiple reaction monitoring (MRM) of glycosaminoglycan disaccharides obtained from treating urine samples with recombinant heparin lyases and chondroitin lyase. This rapid and sensitive method allows the analysis of glycosaminoglycan content and disaccharide composition in urine samples having concentrations 10-to 100-fold lower than those typically analyzed from patients with metabolic diseases, such as mucopolysaccharidosis. The current method facilitates the analysis low (ng/mL) levels of urinary glycosaminoglycans present in healthy individuals and in patients with pathological conditions, such as inflammation and cancers, that can subtly alter glycosaminoglycan content and composition.
Bioengineered heparin is being investigated as a potential substitute for the animal-sourced anticoagulant drug. One step in the current process to prepare bioengineered heparin involves the conversion of N-sulfo heparosan, rich in →4)GlcNS(1→4) GlcA(1→ sequences (where S is sulfo, GlcN is α-D-glucosamine, and GlcA is β-D-glucuronic acid), to a critical intermediate, rich in →4)GlcNS(1→4) IdoA2S(1→ sequences (where S is sulfo and IdoA is α-L-iduronic acid), using 2-O-sulfotransferase (2-OST) and C5 epimerase (C5-epi). Until now, these heparan sulfate biosynthetic enzymes have been expressed in Escherichia coli grown in shake flask culture as fusion proteins. The current study is focused on the high-cell density fed-batch cultivation of recombinant E. coli strains expressing both enzymes. We report the high productivity expression of active 2-OST and C5-epi enzymes of 6.0 and 2.2 mg/gm dry cell weight, respectively.
fermentation; heparin; sulfotransferase; epimerase; recombinant enzymes
Connective tissues diseases (CTDs) are a heterogeneous group of disorders that share certain clinical characteristics and disturbed immunoregulation. Interstitial lung diseases (ILDs), also known as diffuse parenchymal lung diseases, are among the most serious pulmonary complications associated with CTDs. Interleukin 9 (IL-9), IL-4 and interferon γ (IFN-γ) – cytokines with important roles in autoimmune disease – were studied in CTD patients and CTD-ILD patients.
Material and methods
Sixty-one hospitalized untreated CTD patients were recruited, and 20 healthy volunteers were enrolled as controls. The 61 CTD patients were divided into a simple CTD group and a CTD-ILD group, and the plasma protein IL-9, IL-4 and IFN-γ levels were measured by enzyme-linked immunosorbent assay (ELISA).
The results indicate that the serum IL-9 levels were significantly higher in CTD-ILD and simple CTD patients than they were in healthy controls (each p < 0.05) and that the levels were elevated in CTD-ILD patients compared with simple CTD patients (p < 0.05). The IL-4 levels were higher in CTD-ILD patients than they were in the simple CTD patients (p < 0.05) and healthy controls (p < 0.01). In addition, the serum IL-9 levels were negatively correlated with the level of IFN-γ (r2 = 0.34, p = 0.01), the estimated percentage of predicted forced vital capacity (FVC%) (r2 = 0.36, p = 0.00) and the estimated percentage of predicted diffusing capacity (DLCO%) (r2 = 0.27, p = 0.04) and were positively correlated with the IL-4 level (r2 = 0.31, p = 0.01).
Interleukin-9 may play an important role in the pathogenesis of CTD and may contribute to the progression of interstitial lung injury in CTD patients.
interleukin 9; interleukin 4; interferon γ; connective tissues disease; interstitial lung disease
Glycosaminoglycans (GAGs) have numerous applications in the fields of pharmaceuticals, cosmetics, nutraceuticals, and foods. GAGs are also critically important in the developmental biology of all multicellular animals. GAGs were isolated from chicken egg components including yolk, thick egg white, thin egg white, membrane, calcified shell matrix supernatant, and shell matrix deposit. Disaccharide compositional analysis was performed using ultra high-performance liquid chromatography-mass spectrometry. The results of these analyses showed that all four families of GAGs were detected in all egg components. Keratan sulfate was found in egg whites (thick and thin) and shell matrix (calcified shell matrix supernatant and deposit) with high level. Chondroitin sulfates were much more plentiful in both shell matrix components and membrane. Hyaluronan was plentiful in both shell matrix components and membrane, but were only present in a trace of quantities in the yolk. Heparan sulfate was plentiful in the shell matrix deposit but was present in a trace of quantities in the egg content components (yolk, thick and thin egg whites). Most of the chondroitin and heparan sulfate disaccharides were present in the GAGs found in chicken eggs with the exception of chondroitin and heparan sulfate 2,6-disulfated disaccharides. Both CS and HS in the shell matrix deposit contained the most diverse chondroitin and heparan sulfate disaccharide compositions. Eggs might provide a potential new source of GAGs.
chicken egg; glycosaminoglycans; keratan sulfate; chondroitin sulfate; hyaluronan; heparan sulfate
We validate the utility of ion mobility to measure protein conformational changes induced by the binding of glycosaminoglycan ligands, using the well characterized system of Antithrombin III (ATIII) and Arixtra, a pharmaceutical agent with heparin (Hp) activity. Heparin has been used as a therapeutic anticoagulant drug for several decades through its interaction with ATIII, a serine protease inhibitor that plays a central role in the blood coagulation cascade. This interaction induces conformational changes within ATIII that dramatically enhance the ATIII-mediated inhibition rate. Arixtra is the smallest synthetic Hp containing the specific pentasaccharide sequence required to bind with ATIII. Here we report the first travelling wave ion mobility mass spectrometry (TWIMS) investigation of the conformational changes in ATIII induced by its interaction with Arixtra. Native electrospray ionization mass spectrometry allowed the gentle transfer of the native topology of ATIII and ATIII–Arixtra complex. IM measurements of ATIII and ATIII–Arixtra complex showed a single structure, with well-defined collisional cross section (CCS) values. An average 3.6% increase in CCS of ATIII occurred as a result of its interaction with Arixtra, which agrees closely with the theoretical estimation of the change in CCS based on protein crystal structures. A comparison of the binding behavior of ATIII under both denaturing and non-denaturing conditions confirmed the significance of a folded tertiary structure of ATIII for its biological activity. A Hp oligosaccharide whose structure is similar to Arixtra but missing the 3-O sulfo group on the central glucosamine residue showed a dramatic decrease in binding affinity towards ATIII, but no change in the mobility behavior of the complex, consistent with prior studies that suggested that 3-O sulfation affects the equilibrium constant for binding to ATIII, but not the mode of interaction. In contrast, nonspecific binding by a Hp tetrasaccharide showed more complex mobility behavior, suggesting more promiscuous interactions with ATIII. The effect of collisional activation of ATIII and ATIII–Arixtra complex were also assessed, revealing that the binding of Arixtra provided ATIII with additional stability against unfolding. Overall, our results validate the capability of TWIMS to retain the significant features of the solution structure of a protein–carbohydrate complex so that it can be used to study protein conformational changes induced by the binding of glycosaminoglycan ligands.
The thermal instability of the anticoagulant heparin is associated, in part, with the solvolytic loss of N-sulfo groups. This study describes a new method to assess the increased content of unsubstituted amino groups present in thermally-stressed and autoclave-sterilized heparin formulations. N-acetylation of heparin samples with acetic anhydride-d6 is followed by exhaustive heparinase treatment, and disaccharide analysis by hydrophilic interaction chromatography mass spectrometry. The introduction of stable isotopic label provides a sensitive probe for the detection and localization of the lost N-sulfo groups potentially providing valuable insights into degradation mechanism and the reasons for anticoagulant potency loss.
heparin; sulfate; stability assay; amino group; mass spectrometry
Autophagy plays a complicated role in tumorigenesis in a variety of cancers. Recently, many studies report that some autophagy-related markers could be detected in several types of human tumors. However, fewer studies have been conducted to evaluate the relationship between autophagy and lung cancer, especially in non-small cell lung cancer (NSCLC). In this study, the expression levels of autophagy-related markers Beclin 1 and p62 were detected by Western blot analysis and cell immunofluorescence staining in three human NSCLC cell lines A549, H1299 and HCC827. Then, tissue microarray and immunohistochemical staining were used to determine Beclin 1 and p62 expression in 104 NSCLC specimens collected from patients. Beclin 1 and p62 were observed to primarily distribute in the cytoplasm of the cells. Beclin 1 was expressed more predominantly in male and heavy-smoker and its expression was significantly correlated with the differentiation and lymph node metastasis. p62 expression was negatively correlated with TNM stage and lymph node metastasis. Univariate Cox regression analysis revealed that low expression of Beclin 1 and high expression of p62 were significantly associated with shorter survival. Meanwhile, multivariate Cox regression analysis indicated that Beclin 1 and p62 were independent risk factors related to overall survival for patients with NSCLC. Collectively, our study suggests that Beclin 1 and p62 could serve as potential indicators for the prognosis of patients with NSCLC.
Beclin 1; p62; non-small cell lung cancer; prognosis; autophagy
The stereochemistry of the hexuronic acid residues of the structure of glycosaminoglycans (GAGs) is a key feature that affects their interactions with proteins and other biological functions. Electron based tandem mass spectrometry methods, in particular electron detachment dissociation (EDD), have been able to distinguish glucuronic (GlcA) from iduronic acid (IdoA) residues in some heparan sulfate tetrasaccharides by producing epimer-specific fragments. Similarly, the relative abundance of glycosidic fragment ions produced by collision induced dissociation (CID) or EDD have been shown to correlate with the type of hexuronic acid present in chondroitin sulfate (CS) GAGs. The present work examines the effect of charge state and the degree of sodium cationization on the CID fragmentation products that can be used to distinguish GlcA and IdoA containing chondroitin sulfate A (CSA) and dermatan sulfate (DS) chains. The cross-ring fragments 2,4An and 0,2Xn formed within the hexuronic acid residues are highly preferential for chains containing GlcA, distinguishing it from IdoA. The diagnostic capability of the fragments requires the selection of a molecular ion and fragment ions with specific ionization characteristics, namely charge state and the number of ionizable protons. The ions with the proper characteristics display diagnostic properties for all the CS and DS chains (dp4-dp10) studied.
A capillary zone electrophoresis-laser induced fluorescence detection (CZE-LIF) method was developed for the simultaneous analysis of disaccharides derived from heparan sulfate, chondroitin sulfate/dermatan sulfate, hyaluronan and keratan sulfate. Glycosaminoglycans (GAGs) were first depolymerized with the mixture of GAG lyases (heparinase I, II, III and chondroitinase ABC and chondroitinase AC II) and GAG endohydrolase (keratinase II) and the resulting disaccharides were derivatized by reductive amination with 2-aminoacridone. Nineteen fluorescently labeled disaccharides were separated using 50 mM phosphate buffer (pH 3.3) under reversed polarity at 25 kV. Using these conditions, all the disaccharides examined were baseline-separated in less then 25 min. This CZE-LIF method gave good reproducibility both migration time (≤ 1.03% for intra-day and ≤ 4.4% for inter-day) and the peak area values (≤ 5.6% for intra- and ≤ 8.69% for inter-day). This CZE-LIF method was used for profiling and quantification of GAG derivative disaccharides in bovine cornea. The results shows that the current CZE-LIF method offers fast, simple, sensitive, reproducible determination of disaccharides derived from total GAGs in a single run.
Capillary electrophoresis; biopolymers; enzymes
Glycosaminoglycans (GAGs) are a class of biologically important molecules and their structural analysis is the target of considerable research effort. Advances in tandem mass spectrometry (MS/MS) have recently enabled the structural characterization of several classes of GAGs. However, the highly sulfated GAGs, such as heparins, have remained a relatively intractable class due their tendency to lose SO3 during MS/MS producing few sequence-informative fragment ions. The present work demonstrates for the first time the complete structural characterization of the highly sulfated heparin-based drug, Arixtra. This was achieved by Na+/H+ exchange, to create a more ionized species that was stable against SO3 loss, and that produced complete sets of both glycosidic and cross-ring fragment ions. MS/MS, enabling the complete structural determination of Arixtra®, including the stereochemistry of its uronic acid residues and suggests an approach for solving the structure of more complex, highly sulfated heparin-based drugs.
Complete heparin digestion with heparin lyase 2 affords a mixture of disaccharides and resistant tetra-saccharides with 3-O-sulfo group containing glucosamine residues at their reducing ends. Quantitative on-line liquid chromatography-mass spectrometric analysis of these resistant tetrasaccharides is described. The disaccharide and tetrasaccharide composition of seven porcine intestinal heparins and five low molecular weight heparins were analyzed by this method. These resistant tetrasaccharides account for from 5.3 to 7.3 wt% of heparin and from 6.2 to 8.3 wt% of low molecular weight heparin. Since these tetrasaccharides are derived from heparin’s antithrombin III-binding sites, we examined whether this method could be applied to estimate the anticoagulant activity of heparin. The content of 3-O-sulfo group containing tetrasaccharides in a heparin positively correlated (r = 0.8294) to heparin’s anticoagulant activity.
3-O-sulfo group; tetrasaccharides; porcine intestinal heparin; anticoagulant activity; RPIP-LC-MS; heparin lyase 2
molecular weight heparins (LMWHs) are heterogeneous, polydisperse,
and highly negatively charged mixtures of glycosaminoglycan chains
prescribed as anticoagulants. The detailed characterization of LMWH
is important for the drug quality assurance and for new drug research
and development. In this study, online hydrophilic interaction chromatography
(HILIC) Fourier transform mass spectrometry (FTMS) was applied to
analyze the oligosaccharide fragments of LMWHs generated by heparin
lyase II digestion. More than 40 oligosaccharide fragments of LMWH
were quantified and used to compare LMWHs prepared by three different
manufacturers. The quantified fragment structures included unsaturated
disaccharides/oligosaccharides arising from the prominent repeating
units of these LMWHs, 3-O-sulfo containing tetrasaccharides
arising from their antithrombin III binding sites, 1,6-anhydro ring-containing
oligosaccharides formed during their manufacture, saturated uronic
acid oligosaccharides coming from some chain nonreducing ends, and
oxidized linkage region oligosaccharides coming from some chain reducing
ends. This bottom-up approach provides rich detailed structural analysis
and quantitative information with high accuracy and reproducibility.
When combined with the top-down approach, HILIC LC-FTMS based analysis
should be suitable for the advanced quality control and quality assurance
in LMWH production.
Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders, but impurities, structural heterogeneity or functional irreversibility can limit treatment options. We report a series of synthetic LMWHs prepared by cost-effective chemoenzymatic methods. The high activity of one defined synthetic LMWH against human factor Xa (FXa) was reversible in vitro and in vivo using protamine, demonstrating that synthetically accessible constructs can have a critical role in the next generation of LMWHs.
chemoenzymatic synthesis of heparan sulfate tetrasaccharide
(1) and hexasaccharide (2) with a fluorous
tag attached at the reducing end is reported. The fluorous tert-butyl dicarbonate (FBoc) tag did not interfere
with enzymatic recognition for both elongation and specific sulfation,
and flash purification was performed by standard fluorous solid-phase
extraction (FSPE). Based on an FBoc attached disaccharide
as acceptor, a series of partial N-sulfated, 6-O-sulfated heparan sulfate oligosaccharides were successfully
synthesized employing fluorous techniques.
Glycans play a critical role in physiological and pathological processes through interaction with a variety of ligands. Altered expression and dysregulation of these molecules can cause aberrant cellular function such as malignancy. Glycomics provide information of the structure and function of glycans, glycolipids, and glycoproteins such as proteoglycans, and may help to predict cancer development and progression as biomarkers. In this report, we compared the expression of proteoglycans, the content and structure of glycosaminoglycans and glycolipids between patient-matched normal and cancer tissues obtained from colon cancer patients. Tumor-related proteoglycans, glypican-3, and syndecan-1 showed downregulation in cancer tissues compared to normal tissues. In cancer tissue, the total amount of chondroitin sulfate (CS)/dermatan sulfate and heparan sulfate were lower and, interestingly, the level of disaccharide units of both 4S6S (CS-E) and 6S (CS-C) were higher compared to normal tissue. Also, overall lipids including glycolipids, a major glycomics target, were analyzed by hydrophilic interaction liquid chromatography mass spectrometry. Increase of lyso-phosphatidylcholine (phospholipid), sphingomyelin (sphigolipid), and four types of glycolipids (glucosylceramide, lactosylceramide, monosialic acid ganglioside, and globoside 4) in cancer tissue showed the possibility as potential biomarkers in colon cancer. While requiring the need for careful interpretation, this type of broad investigation gives us a better understanding of pathophysiological roles on glycosaminoglycans and glycolipids and might be a powerful tool for colon cancer diagnosis.
High throughput (HT) platforms serve as cost-efficient and rapid screening method for evaluating the effect of cell culture conditions and screening of chemicals. The aim of the current study was to develop a high-throughput cell-based microarray platform to assess the effect of culture conditions on Chinese hamster ovary (CHO) cells. Specifically, growth, transgene expression and metabolism of a GS/MSX CHO cell line, which produces a therapeutic monoclonal antibody, was examined using microarray system in conjunction with conventional shake flask platform in a non-proprietary medium. The microarray system consists of 60 nl spots of cells encapsulated in alginate and separated in groups via an 8-well chamber system attached to the chip. Results show the non-proprietary medium developed allows cell growth, production and normal glycosylation of recombinant antibody and metabolism of the recombinant CHO cells in both the microarray and shake flask platforms. In addition, 10.3 mM glutamate addition to the defined base media results in lactate metabolism shift in the recombinant GS/MSX CHO cells in the shake flask platform. Ultimately, the results demonstrate that the high-throughput microarray platform has the potential to be utilized for evaluating the impact of media additives on cellular processes, such as, cell growth, metabolism and productivity.
cell-based microarray; high through-put; CHO cells; media optimization; 3D cell culture
Heparin is a critically important anticoagulant drug that was contaminated with a persulfonated polysaccharide in 2008, resulting in a number of severe adverse reactions, some leading to death. Controversy remains as to the precise composition of the 2008 contaminant and new information suggests that heparin may now be subject to adulteration with a new, difficult to detect, contaminant, N-sulfo oversulfated chondroitin sulfate. This study synthesizes this new potential contaminant and describes the use of radical depolymerization followed by liquid chromatography-mass spectrometry to detect N-sulfo oversulfated chondroitin sulfate and to confirm the structure of the 2008 contaminant as oversulfated chondroitin sulfate and not oversulfated heparan sulfate.
heparin; oversulfated chondroitin sulfate; oversulfated heparan sulfate; N-sulfated oversulfated chondroitin sulfate; contaminant; radical depolymerization; mass spectrometry
O-sulfotransferases (OSTs) are critical enzymes in the cellular biosynthesis of the biologically and pharmacologically important heparan sulfate and heparin. Recently, these enzymes have been cloned and expressed in bacteria for application in the chemoenzymatic synthesis of glycosaminoglycan-based drugs. OST activity assays have largely relied on the use of radioisotopic methods using [35S] 3'-phosphoadenosine-5'-phosphosulfate and scintillation counting. Herein, we examine alternative assays that are more compatible with a biomanufacturing environment. A high throughput microtiter-based approach is reported that relies on a coupled bienzymic colorimetric assay for heparan sulfate and heparin OSTs acting on polysaccharide substrates using arylsulfotransferase-IV and p-nitrophenylsulfate as a sacrificial sulfogroup donor. A second liquid chromatography-mass spectrometric assay, for heparan sulfate and heparin OSTs acting on structurally defined oligosaccharide substrates, is also reported that provides additional information on the number and positions of the transferred sulfo groups within the product. Together, these assays allow quantitative and mechanistic information to be obtained on OSTs that act on heparan sulfate and heparin precursors.
enzymes; mass spectrometry; bioassays; sulfotransferases; coupled assay; heparin; heparan sulfate
This paper presents a density functional theory (DFT)/time-dependent DFT (TD-DFT) study on the lowest lying singlet and triplet excited states of 20 selected polybrominateddiphenyl ether (PBDE) congeners, with the solvation effect included in the calculations using the polarized continuum model (PCM). The results obtained showed that for most of the brominated diphenyl ether (BDE) congeners, the lowest singlet excited state was initiated by the electron transfer from HOMO to LUMO, involving a π–σ* excitation. In triplet excited states, structure of the BDE congeners differed notably from that of the BDE ground states with one of the specific C–Br bonds bending off the aromatic plane. In addition, the partial least squares regression (PLSR), principal component analysis-multiple linear regression analysis (PCA-MLR), and back propagation artificial neural network (BP-ANN) approaches were employed for a quantitative structure-property relationship (QSPR) study. Based on the previously reported kinetic data for the debromination by ultraviolet (UV) and sunlight, obtained QSPR models exhibited a reasonable evaluation of the photodebromination reactivity even when the BDE congeners had same degree of bromination, albeit different patterns of bromination.
Polybrominateddiphenyl ethers; theoretical study; excited states; photodebromination; quantitative structure-property relationship; artificial neural network