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.     

Pentraxin 3 (PTX3) Is Associated with Severe Sepsis and Fatal Disease in Emergency Room Patients with Suspected Infection: A Prospective Cohort Study

Background

Early diagnostic and prognostic stratification of patients with suspected infection is a difficult clinical challenge. We studied plasma pentraxin 3 (PTX3) upon admission to the emergency department in patients with suspected infection.

Methods

The study comprised 537 emergency room patients with suspected infection: 59 with no systemic inflammatory response syndrome (SIRS) and without bacterial infection (group 1), 67 with bacterial infection without SIRS (group 2), 54 with SIRS without bacterial infection (group 3), 308 with sepsis (SIRS and bacterial infection) without organ failure (group 4) and 49 with severe sepsis (group 5). Plasma PTX3 was measured on admission using a commercial solid-phase enzyme-linked immunosorbent assay (ELISA).

Results

The median PTX3 levels in groups 1–5 were 2.6 ng/ml, 4.4 ng/ml, 5.0 ng/ml, 6.1 ng/ml and 16.7 ng/ml, respectively (p<0.001). The median PTX3 concentration was higher in severe sepsis patients compared to others (16.7 vs. 4.9 ng/ml, p<0.001) and in non-survivors (day 28 case fatality) compared to survivors (14.1 vs. 5.1 ng/ml, p<0.001). A high PTX3 level predicted the need for ICU stay (p<0.001) and hypotension (p<0.001). AUC^ROC in the prediction of severe sepsis was 0.73 (95% CI 0.66–0.81, p<0.001) and 0.69 in case fatality (95% CI 0.58–0.79, p<0.001). PTX3 at a cut-off level for 14.1 ng/ml (optimal cut-off value for severe sepsis) showed 63% sensitivity and 80% specificity. At a cut-off level 7.7 ng/ml (optimal cut-off value for case fatality) showed 70% sensitivity and 63% specificity in predicting case fatality on day 28.In multivariate models, high PTX3 remained an independent predictor of severe sepsis and case fatality after adjusting for potential confounders.

Conclusions

A high PTX3 level on hospital admission predicts severe sepsis and case fatality in patients with suspected infection.     

Predator-induced synchrony in population oscillations of coexisting small mammal species

Comprehensive analyses of long-term (1977-2003) small-mammal abundance data from western Finland showed that populations of Microtus voles (field voles M. agrestis and sibling voles M. rossiaemeridionalis) voles, bank (Clethrionomys glareolus) and common shrews (Sorex araneus) fluctuated synchronously in 3 year population cycles. Time-series analyses indicated that interspecific synchrony is influenced strongly by density-dependent processes. Synchrony among Microtus and bank voles appeared additionally to be influenced by density-independent processes. To test whether interspecific synchronization through density-dependent processes is caused by predation, we experimentally reduced the densities of the main predators of small mammals in four large agricultural areas, and compared small mammal abundances in these to those in four control areas (2.5-3 km(2)) through a 3 year small-mammal population cycle. Predator reduction increased densities of the main prey species, Microtus voles, in all phases of the population cycle, while bank voles, the most important alternative prey of predators, responded positively only in the low and the increase phase. Manipulation also increased the autumn densities of water voles (Arvicola terrestris) in the increase phase of the cycle. No treatment effects were detected for common shrews or mice. Our results are in accordance with the alternative prey hypothesis, by which predators successively reduce the densities of both main and alternative prey species after the peak phase of small-mammal population cycles, thus inducing a synchronous low phase.     

From the subarctic to the tropics: effects of 4-month deployment on soldiers' heat stress, heat strain, and physical performance

The aim of the study was to evaluate the heat stress of Finnish male soldiers (N = 20, age 22.0 ± 2.5 years, body mass 78.8 ± 11.5 kg, and height 180.2 ± 5.6 cm) during their 4-month deployment in a hot environment and to find out the effects on physical performance and body composition. The troops moved from 2.5° C (mean monthly temperature) in Finland to 31.9° C in Chad. During the deployment, temperatures varied between 13.5 and 57.0° C outdoors and in the vehicles and tents. During 1-day recording in the middle of the deployment, skin temperatures were 34-35° C during daytime and maximal core temperature remained mainly below 38.0° C. Body mass decreased (78.4 ± 11.5 kg vs. 75.6 ± 8.6, p = 0.007) during the deployment without changes in fat mass. The sit-up performance increased by 10.9% (46 ± 10 reps·min?1 vs. 51 ± 7 reps·min?1, p < 0.01), and the maximal force production of the leg extensor muscles increased (3,042 ± 614 N vs. 3,277 ± 706 N, p < 0.05) without change in the rate of force development. No changes were observed in the push-ups, repeated squats, maximal grip strength, and running distance during the 12-minute test. In conclusion, the soldiers were able to maintain or improve their physical performance during the deployment despite the heat stress. It is important to encourage soldiers to engage in physical training, especially during a thermally appropriate time of the day or in air-conditioned facilities. Monitoring of local heat stress is also recommended.     

A dual-step fluorescence resonance energy transfer-based quenching assay for screening of caspase-3 inhibitors

The control of cell death is an intricate process involving a multitude of intracellular modulators. Among these molecules, the caspases have a central role and have become an interesting group of enzymes in the current pharmaceutical industry. We have developed a novel dual-step fluorescence energy transfer-based separation-free assay method for the primary screening of caspase-3 inhibitors in vitro. This method relies on fluorescent europium(III)-chelate-doped nanoparticle donors coated with streptavidin in conjunction with a dual-labeled (N-terminal Alexa Fluor 680 fluorescent acceptor and C-terminal BlackBerry Quencher 650) caspase-3-specific peptide substrate modified with a biotinyl moiety. In the assay, the nanoparticle donor excites the fluorescent acceptor, whose emission is monitored with time-resolved measurements. The intensity of the acceptor reflects the activity of the enzyme because the intensity is controlled by the proximity of the quencher. Owing to the dual-step fluorescence resonance energy transfer, this method enables a sensitized fluorescence signal directly proportional to the extent of enzymatic activity with relatively background fluorescence-free measurements in the event of complete enzyme inhibition. The generic nanoparticle donors further promote versatility and cost-efficiency of the method. The performance evaluated as the inhibitor (Z-DEVD-FMK) dose-response curve (IC(50) value of approximately 12 nM) was in good agreement with that of the recent methods found in literature. This assay serves as a model application proving the feasibility of the europium-chelate-doped nanoparticle labels in a homogeneous assay for proteolytic activity.     

Reproducibility-optimized test statistic for ranking genes in microarray studies

A principal goal of microarray studies is to identify the genes showing differential expression under distinct conditions. In such studies, the selection of an optimal test statistic is a crucial challenge, which depends on the type and amount of data under analysis. While previous studies on simulated or spike-in datasets do not provide practical guidance on how to choose the best method for a given real dataset, we introduce an enhanced reproducibility-optimization procedure, which enables the selection of a suitable gene- anking statistic directly from the data. In comparison with existing ranking methods, the reproducibilityoptimized statistic shows good performance consistently under various simulated conditions and on Affymetrix spike-in dataset. Further, the feasibility of the novel statistic is confirmed in a practical research setting using data from an in-house cDNA microarray study of asthma-related gene expression changes. These results suggest that the procedure facilitates the selection of an appropriate test statistic for a given dataset without relying on a priori assumptions, which may bias the findings and their interpretation. Moreover, the general reproducibilityoptimization procedure is not limited to detecting differential expression only but could be extended to a wide range of other applications as well.     

Crowding-induced responses in a geometrid moth revisited: a field experiment

In Lepidoptera, effects of larval crowding on life history traits may manifest themselves as changes in growth rate and duration of larval period. For the outbreaking geometrid moth, Epirrita autumnata , impacts of crowding have been shown in earlier laboratory studies, and these responses were modified by diet quality. In this study, the aim was to find out the specific nature of crowding effects of E. autumnata in the field. E. autumnata larvae were reared individually and in groups in mesh bags on mountain birch, both on good and poor quality foliage. Three field experiments were carried out with different densities in the crowded treatment (12, 21 and 45 larvae per 100 short shoots of mountain birch), simulating intermediate to severe outbreaks. The study revealed the density interval (12–21) within which impacts of crowding become evident. Significant effects were mostly found with the two highest densities in the crowded treatment combined with poor foliage quality. In those treatments, crowding resulted in 8–12% and 24–33% decrease in pupal mass and larval survival, respectively. The results of the present field experiments, however, did not corroborate the results of earlier laboratory studies: duration of larval period was not substantially affected, net effects of crowding were negative and interactions between crowding and foliage quality were contrary to those obtained in earlier laboratory studies. In many other Lepidoptera as well, the knowledge on responses to crowding may be mostly qualitative since most crowding experiments have been conducted in laboratory.     

Resonance assignments of the 56 kDa chimeric avidin in the biotin-bound and free forms

Avidin is a homotetrameric ~56 kDa protein found in chicken egg white. Avidin’s ability to bind biotin with a very high affinity has widely been exploited in biotechnological applications. Protein engineering has further diversified avidin’s feasibility. ChiAVD(I117Y) is a product of rational protein engineering. It is a hyperthermostable synthetic hybrid of avidin and avidin-related protein 4 (AVR4). In this chimeric protein a 23-residue segment in avidin has been replaced with the corresponding sequence found in AVR4, and a point mutation at subunit interface 1–3 (and 2–4) has been introduced. Here we report the backbone and sidechain resonance assignments of the biotin-bound form of ChiAVD(I117Y) as well as the backbone resonance assignments of the free form.     

Homogeneous TR-FRET high-throughput screening assay for calcium-dependent multimerization of sorcin

A homogeneous high-throughput screening method based on time-resolved fluorescence resonance energy transfer (TR-FRET) for the measurement of calcium-dependent multimerization of an EF-hand protein, sorcin, is described. The assay is based on a specific sorcin binding peptide conjugated either with an intrinsically highly fluorescent europium chelate (donor) or an Alexa Fluor 700 fluorophore (acceptor). Addition of calcium results in multimerization of sorcin, allowing several peptides to bind simultaneously to the epitopes of the multimeric protein complex, and the proximity of peptides labeled either with donor or acceptor label results in fluorescence resonance energy transfer between the 2 labels. When no calcium is present, the protein remains in a monomer form, and thus no FRET can take place. In the optimized assay construct, the assay was performed in 45 min, and a more than 20-fold signal-to-background ratio was achieved. The reversibility of sorcin multimerization was shown by chelating free calcium with ethylenediamine tetraacetic acid (EDTA). The developed homogeneous assay can be used in screening molecules that either inhibit or enhance multimerization of sorcin, and the assay format is applicable to various noncompetitive high-throughput screening assays detecting protein multimerization reactions.     

Delayed density-dependent parasitism of eggs and pupae as a contributor to the cyclic population dynamics of the autumnal moth

Many populations of forest Lepidoptera exhibit 10-year cycles in densities, with impressive outbreaks across large regions. Delayed density-dependent interactions with natural enemies are recognized as key factors driving these cyclic population dynamics, but emphasis has typically been on the larval stages. Eggs, pupae and adults also suffer mortality from predators, parasitoids and pathogens, but little is known about possible density relationships between mortality factors and these non-feeding life stages. In a long-term field study, we experimentally deployed autumnal moth (Epirrita autumnata) eggs and pupae to their natural enemies yearly throughout the 10-year population cycle in northern Norway. The abundance of another geometrid, the winter moth (Operophtera brumata), increased in the study area, permitting comparisons between the two moth species in predation and parasitism. Survival of autumnal moth eggs and pupae was related to the moth abundance in an inverse and delayed manner. Egg and pupal parasitoids dominated as density-dependent mortality factors and predicted the subsequent growth rate of the host population size. In contrast, effects of egg and pupal predators were weakly density dependent, and generally predation remained low. Parasitism rates did not differ between the autumnal and winter moth pupae, whereas predators preferred winter moth pupae over those of the autumnal moth. We conclude that parasitism of the autumnal moth by egg and pupal parasitoids can be related to the changes of the moth density in a delayed density-dependent manner. Furthermore, egg and pupal parasitoids cannot be overlooked as causal factors for the population cycles of forest Lepidoptera in general.