N-glycomic Profiling as a Tool to Separate Rectal Adenomas from Carcinomas

All human cells are covered by glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans. Most glycans are localized to cell surfaces and participate in events essential for cell viability and function. Glycosylation evolves during carcinogenesis, and therefore carcinoma-related glycan structures are potential cancer biomarkers. Colorectal cancer is one of the world''s three most common cancers, and its incidence is rising. Novel biomarkers are essential to identify patients for targeted and individualized therapy. We compared the N-glycan profiles of five rectal adenomas and 18 rectal carcinomas of different stages by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Paraffin-embedded tumor samples were deparaffinized, and glycans were enzymatically released and purified. We found differences in glycosylation between adenomas and carcinomas: monoantennary, sialylated, pauci-mannose, and small high-mannose N-glycan structures were more common in carcinomas than in adenomas. We also found differences between stage I–II and stage III carcinomas. Based on these findings, we selected two glycan structures: pauci-mannose and sialyl Lewis a, for immunohistochemical analysis of their tissue expression in 220 colorectal cancer patients. In colorectal cancer, poor prognosis correlated with elevated expression of sialyl Lewis a, and in advanced colorectal cancer, poor prognosis correlated with elevated expression of pauci-mannose. In conclusion, by mass spectrometry we found several carcinoma related glycans, and we demonstrate a method of transforming these results into immunohistochemistry, a readily applicable method to study biomarker expression in patient samples.Glycans, the carbohydrate units of glycoproteins, glycolipids, and proteoglycans, that cover all human cells. Around 1% of the human genome participates in the biosynthesis of glycans(1). This biosynthesis is the most complex post-translational modification of proteins, and the great variability in glycan structures contains a tremendous ability to fine-tune the chemical and biological properties of glycoproteins. The glycosylation process occurs most abundantly in the Golgi apparatus and the endoplasmic reticulum, but also occurs in the cytoplasm and nucleus (2). Most glycoconjugates are localized to cell surfaces, where glycans participate in events essential for cell viability and function, such as cell adhesion, motility, and intracellular signaling (2). Changes in these functions are key steps seen when normal cells transform to malignant ones, and these are also reflected in changes of a cell''s glycan profile, observed in many cancers (3, 4). Specific structural changes in glycans may serve as cancer biomarkers (5, 6), and changes in glycosylation profiles are related to aggressive behavior in tumor cells (7–9).Cancer-associated asparagine-linked glycan (N-glycan) structures may play specific roles in supporting tumor progression; growth (10, 11), invasion (12, 13), and angiogenesis (14). Changes in the N-glycan profile emerge in numerous cancers, including lung (15, 16), breast (17), and colorectal cancer (CRC)¹ (16, 18). Balog et al. (18) comparing the N-glycomic profile of CRC tissue to adjacent normal mucosa, reported differences in specific glycan structures. Moreover, serum N-glycosylation profile from patients with CRC differ from those of healthy controls (19).Colorectal cancer is the third most common cause of cancer-related death worldwide and its incidence is rising; 40% of CRCs are of rectal origin. Roughly 40% of patients have localized disease (stage I–II; Dukes A–B), another 40% loco regional disease (stage III; Dukes C), and 20% metastasized disease (stage IV; Dukes D) (20). Although stage at diagnosis is the most important factor determining prognosis, clinical outcome, and response to adjuvant treatment can markedly vary within each stage. Adjuvant therapy routinely goes to stage III patients, but the benefit of adjuvant treatment for stage II patients is unclear. Of stage II patients, 80% are cured by radical surgery alone. To identify patients who will benefit from postoperative treatment, we need novel biomarkers. The glycan profile of the tumor tissue could provide new biomarkers for diagnosis and prognosis of cancer.In this study, we characterized the N-glycomic profiles of rectal adenomas and carcinomas by MALDI-TOF mass spectrometric (MS) profiling of asparagine-linked glycans. Our aim was to identify differences between adenomas and carcinomas, and also between cancers of different stages. Based on glycan profiling, we also chose, for immunohistochemical expression studies of a series of 220 CRC patients, two glycan markers: sialyl Lewis a and pauci-mannose.     

Scaffold-hopping with zwitterionic CCR3 antagonists: Identification and optimisation of a series with good potency and pharmacokinetics leading to the discovery of AZ12436092

The discovery and optimisation of a series of zwitterionic CCR3 antagonists is described. Optimisation of the structure led to AZ12436092, a compound with excellent selectivity over activity at hERG and outstanding pharmacokinetics in preclinical species.     

Do warmer growing seasons ameliorate the recovery of mountain birches after winter moth outbreak?

Subarctic mountain birch (Betula pubescens ssp. czerepanovii) forests in northern Fennoscandia have shown a slight recovery from recent severe defoliation by the winter moth (Operophtera brumata). This development in trees is hypothesized to be a result of ameliorated growing conditions through increased summer temperatures. We examined if accumulated thermal sum affects the ability of mountain birches to tolerate foliage losses. We quantified the number of leaf-bearing short shoots, the emergence of inflorescences and the seasonal height growth of long shoots in both intact and defoliated trees. We also determined the concentrations of carbon and nitrogen in leaves and carbohydrates in roots. Our results show that defoliation constrained the growth of long shoots, as well as the emergence of inflorescences regardless of thermal sum accumulation. However, the number of leaf-bearing short shoots did not differ between intact and defoliated trees. In the both tree groups, the amounts of emerging leaves increased as a response to thermal sum accumulation. Also the leaf carbon concentration increased in defoliated trees at higher thermal sums, whereas it decreased in intact controls. Generally, the mean carbohydrate concentrations were greater in roots of defoliated than intact trees. However, with increased thermal sums, root carbohydrates increased in intact trees but remained the same in defoliated trees. We conclude that thermal sum accumulation does not greatly promote the recovery of mountain birches. Although the damaged trees produced more leaves at warmer growing sites, this did not increase their height growth or carbohydrate gain in roots.     

Glycomics of bone marrow-derived mesenchymal stem cells can be used to evaluate their cellular differentiation stage

Human mesenchymal stem cells (MSCs) are adult multipotent progenitor cells. They hold an enormous therapeutic potential, but at the moment there is little information on the properties of MSCs, including their surface structures. In the present study, we analyzed the mesenchymal stem cell glycome by using mass spectrometric profiling as well as a panel of glycan binding proteins. Structural verifications were obtained by nuclear magnetic resonance spectroscopy, mass spectrometric fragmentation, and glycosidase digestions. The MSC glycome was compared to the glycome of corresponding osteogenically differentiated cells. More than one hundred glycan signals were detected in mesenchymal stem cells and osteoblasts differentiated from them. The glycan profiles of MSCs and osteoblasts were consistently different in biological replicates, indicating that stem cells and osteoblasts have characteristic glycosylation features. Glycosylation features associated with MSCs rather than differentiated cells included high-mannose type N-glycans, linear poly-N-acetyllactosamine chains and α2-3-sialylation. Mesenchymal stem cells expressed SSEA-4 and sialyl Lewis x epitopes. Characteristic glycosylation features that appeared in differentiated osteoblasts included abundant sulfate ester modifications. The results show that glycosylation analysis can be used to evaluate MSC differentiation state.     

Development of a denaturation/renaturation-based production process for ferritin nanoparticles

Recently, we presented a simple method for generating biological functional protein-based nanoparticles that are ready for use as label agents in bioaffinity assays (J??skel?inen et al., 2007 Small 3:1362-1367). In this process, the particle shell (ferritin protein) and binding molecules are conjugated via genetic fusion, and particles with binding capacity are produced in a single bacterial cultivation. Production is combined with simple, non-chromatographic purification during which Europium ions are introduced into particles to serve as marker agents. Denaturation-refolding has previously performed by means of pH changes. Here, we test urea as an alternative agent for denaturation, and examine techniques to improve refolding of the functional particles. Three different types of binding molecules were employed in our experiments: biotin carboxyl carrier protein (a small protein with 87 amino acids), single chain antibody fragment (a complex binding protein) and calmodulin-binding peptide (27 amino acids). Urea was successfully utilized to generate functional particles with inherent binding activity and label function. Additionally, particle yield was effectively optimized by analyzing various refolding and bacterial production conditions. Our results clearly demonstrate that this simple biological method of producing functional ferritin-based particles is flexible, and different types of binding moieties can be applied by adjusting the production conditions.     

Genome-Wide Scoring of Positive and Negative Epistasis through Decomposition of Quantitative Genetic Interaction Fitness Matrices

Recent technological developments in genetic screening approaches have offered the means to start exploring quantitative genotype-phenotype relationships on a large-scale. What remains unclear is the extent to which the quantitative genetic interaction datasets can distinguish the broad spectrum of interaction classes, as compared to existing information on mutation pairs associated with both positive and negative interactions, and whether the scoring of varying degrees of such epistatic effects could be improved by computational means. To address these questions, we introduce here a computational approach for improving the quantitative discrimination power encoded in the genetic interaction screening data. Our matrix approximation model decomposes the original double-mutant fitness matrix into separate components, representing variability across the array and query mutants, which can be utilized for estimating and correcting the single-mutant fitness effects, respectively. When applied to three large-scale quantitative interaction datasets in yeast, we could improve the accuracy of scoring various interaction classes beyond that obtained with the original fitness data, especially in synthetic genetic array (SGA) and in genetic interaction mapping (GIM) datasets. In addition to the known pairs of interactions used in the evaluation of the computational approach, a number of novel interaction pairs were also predicted, along with underlying biological mechanisms, which remained undetected by the original datasets. It was shown that the optimal choice of the scoring function depends heavily on the screening approach and on the interaction class under analysis. Moreover, a simple preprocessing of the fitness matrix could further enhance the discrimination power of the epistatic miniarray profiling (E-MAP) dataset. These systematic evaluation results provide in-depth information on the optimal analysis of the future, large-scale screening experiments. In general, the modeling framework, enabling accurate identification and classification of genetic interactions, provides a solid basis for completing and mining the genetic interaction networks in yeast and other organisms.     

Does immune function influence population fluctuations and level of parasitism in the cyclic geometrid moth?

Populations of the autumnal moth, Epirrita autumnata, exhibit cycles with high amplitudes in northernmost Europe, culminating in devastating outbreak densities at favourable sites. Parasitism by hymenopteran parasitoids has been hypothesised to operate with a delayed density dependence capable of producing the observed dynamics. It has also been hypothesised that insects in crowded conditions invest greatly in their immunity as a counter-measure to increased risk of parasitism and pathogen infections. Furthermore, inducible plant defences consequent to grazing by herbivorous insects may be linked to the performance of parasitoids and pathogens through increased immunocompetence of the herbivore feeding on the foliage, in which the defence induction has taken place. At ten sampling sites, we quantified larval abundance, outbreak status and percentage larval parasitism during an extended peak phase of a population cycle. These population level covariates, together with an individual pupal mass, were used to explain differences in the immune defence, measured as an encapsulation reaction to artificial antigen. We also conducted a field study for an investigation of the susceptibility of autumnal moth pupae to naturally occurring pupal parasitoids. We did not find obvious differences between the encapsulation rate of autumnal moths originating from the sites with different past and current larval densities and risks for parasitism. The best ranked statistical models included pupal mass and outbreak status as explanatory variables, although both showed only slight effects on the encapsulation rate. The host resistance test revealed positive relationships between the encapsulation rate, body size and percentage parasitism of the exposed pupae, indicating that pupal parasitoids chose, and/or survived better, in large host individuals irrespective of their encapsulation ability. Thus, our results do not provide support for the hypothesis that variation in the immune function drives or modulates population cycles of autumnal moths. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Performance of the cyclic autumnal moth,<Emphasis Type="Italic"> Epirrita autumnata</Emphasis>, in relation to birch mast seeding

The mast depression hypothesis has been put forward to explain the 9- to 10-year population cycle of the autumnal moth (Epirrita autumnata; Lepidoptera: Geometridae) in northern Fennoscandia. We analysed long-term data from Finnish Lapland in order to evaluate the critical assumption of the mast depression hypothesis: that better individual performance of herbivores, followed by high annual growth rate of populations, occurs in the year following mast seeding of the host, the mountain birch ( Betula pubescens ssp. czerepanovii). Since mast seeding has been suggested to occur at the expense of chemical defence against herbivores, we bioassayed the quality of birch leaves from the same trees by means of yearly growth trials with autumnal moth larvae. We also measured the size of wild adults as a determinant of potential fecundity of the species in different years. The relative growth rate of larvae was poorer in post-mast years compared to other years, rather than better as assumed by the hypothesis. Conversely, a slight indication of the increase in potential fecundity was observed due to the somewhat larger adult size in post-mast years. Population growth rate estimates, however, showed that the increase in fecundity would have to be much higher to facilitate population increase towards a cyclic peak with outbreak density. Accordingly, our two data sets do not support the assumption of a higher annual growth rate in autumnal moth populations subsequent to mast seeding of the host, thereby contradicting the predictions of the mast depression hypothesis. Temperatures, when indexed by the North Atlantic Oscillation and accumulated thermal sums, were observed to correlate with the abundance or rate of population change of the autumnal moth. The factors underlying the regular population cycles of the autumnal moth, however, remain unidentified. Overall, we suggest that the causal agents in cyclic insect population dynamics should be clarified by field experimentation, since trophic interactions are complex and are further modified by abiotic factors such as climate.     

Reduction in size and fecundity of the autumnal moth, <Emphasis Type="Italic">Epirrita autumnata</Emphasis>, in the increase phase of a population cycle

Increasing fecundity with increasing density has been observed for many cyclic herbivore populations, including some forest Lepidoptera. We monitored population density, body size and reproductive capacity of the cyclic lepidopteran, the autumnal moth (Epirrita autumnata, Geometridae), from the early increase phase to the devastating outbreak density in northernmost Norway. Larval density of the species increased exponentially from 1998 to 2002 and remained at the outbreak level also in 2003. Within the same period, the body size and fecundity of individuals reduced as analysed from several parallel datasets on larvae, pupae and adults. In another study area in northernmost Finland, the density increase of the autumnal moth was moderate only, and true outbreak density was not attained during the study. Despite that, a reduction was again detected in the size and fecundity of individuals. Possible factors responsible for the reduced size and fecundity of individuals in the Norwegian population were quantitative shortage of foliage, rapid and delayed inducible resistances of the host, mountain birch (Betula pubescens ssp. czerepanovii), as well as crowding-induced responses of larvae. These factors likely acted in concert, although non-delayed responses to the density were emphasized. Our findings did not support the hypotheses of climatic release, inducible susceptibility of the host tree and mast depression (i.e. lowered chemical defence of the host tree after its mast seeding) as promoters of the fecundity-based density increase of the autumnal moth, since the reduced fecundity in relation to increased density was strongly against the predictions of these hypotheses. Therefore, we suggest that the density increase of autumnal moth populations is promoted by high survival rather than exceptionally high fecundity.