Isobaric labeling-based quantitative proteomics of FACS-purified immune cells and epithelial cells from the intestine of Crohn's disease patients reveals proteome changes of potential importance in disease pathogenesis

Crohn's disease (CD) is a chronic condition characterized by recurrent flares of inflammation in the gastrointestinal tract. Disease etiology is poorly understood and is characterized by dysregulated immune activation that progressively destroys intestinal tissue. Key cellular compartments in disease pathogenesis are the intestinal epithelial layer and its underlying lamina propria. While the epithelium contains predominantly epithelial cells, the lamina propria is enriched in immune cells. Deciphering proteome changes in different cell populations is important to understand CD pathogenesis. Here, using isobaric labeling-based quantitative proteomics, we perform an exploratory study to analyze in-depth proteome changes in epithelial cells, immune cells and stromal cells in CD patients compared to controls using cells purified by FACS. Our study revealed increased proteins associated with neutrophil degranulation and mitochondrial metabolism in immune cells of CD intestinal mucosa. We also found upregulation of proteins involved in glycosylation and secretory pathways in epithelial cells of CD patients, while proteins involved in mitochondrial metabolism were reduced. The distinct alterations in protein levels in immune- versus epithelial cells underscores the utility of proteome analysis of defined cell types. Moreover, our workflow allowing concomitant assessment of cell-type specific changes on an individual basis enables deeper insight into disease pathogenesis.     

Benzofuranoid neolignans from Licaria armeniaca

The trunkwood of Licaria armeniaca (Nees) Kosterm. (Lauraceae) contains sitosterol, 6,7-dimethoxy-coumarin and two novel benzofuranoid neolignans: (2S, 3S, 3aR, 5R)-3a-allyl-5-methoxy- and 5,7-dimethoxy-2-(3′, 4′-methylenedioxyphenyl)-3-methyl-2,3,3a,4,5,6-hexahydro-6-oxobenzofurans.     

The spatial pattern of DNA synthesis in Dictyostelium discoideum slugs

We have used [³H]DNA labelling and autoradiography to investigate the localisation of cells in S phase of the cell cycle during the aggregation and slug stages of Dictyostelium discoideum development. Our results indicate that S phase cells occur behind a sharp transverse boundary, which falls just below (or behind) the anterior tip of the aggregate or slug. PAS staining indicates that this is the boundary between the prestalk and prespore regions.     

Multiple properties of the splicing repressor SRp38 distinguish it from typical SR proteins

The SR protein SRp38 is a general splicing repressor that is activated by dephosphorylation during mitosis and in response to heat shock. Here we describe experiments that provide insights into the mechanism by which SRp38 functions in splicing repression. We first show that SRp38 redistributes and colocalizes with snRNPs, but not with a typical SR protein, SC35, during mitosis and following heat shock. Supporting the functional significance of this association, a micrococcal nuclease-sensitive component, i.e., an snRNP(s), completely rescued heat shock-induced splicing repression in vitro, and purified U1 snRNP did so partially. SRp38 contains an N-terminal RNA binding domain (RBD) and a C-terminal RS domain composed of two subdomains (RS1 and RS2 domains). Unexpectedly, an RS1 deletion mutant derivative specifically inhibited the second step of splicing, while an RS2 deletion mutant retained significant dephosphorylation-dependent repression activity. Using chimeric SRp38/SC35 proteins, we show that SC35-RBD/SRp38-RS can function as a general splicing activator and that the dephosphorylated version can act as a strong splicing repressor. SRp38-RBD/SC35-RS, however, was essentially inactive in these assays. Together, our results help to define the unusual features of SRp38 that distinguish it from other SR proteins.     

Epicardial Fat from Echocardiography: A New Method for Visceral Adipose Tissue Prediction

Objective: To validate transthoracic echocardiography as an easy and reliable imaging method for visceral adipose tissue (VAT) prediction. VAT is recognized as an important indicator of high cardiovascular and metabolic risk. Several methods are applied to estimate VAT, with different results. Research Methods and Procedures: We selected 60 healthy subjects (29 women, 31 men, 49.5 ± 16.2 years) with a wide range of body mass indexes. Each subject underwent transthoracic echocardiogram and magnetic resonance imaging (MRI) to measure epicardial fat thickness on the right ventricle. Measurements of epicardial adipose tissue thickness were obtained from the same echocardiographic and MRI views and points. MRI was also used to measure VAT cross‐sectional areas at the level of L4 to L5. Anthropometric indexes were also measured. Results: Subjects with predominant visceral fat accumulation showed higher epicardial adipose tissue thickness than subjects with predominant peripheral fat distribution: 9.97 ± 2.88 vs. 4.34 ± 1.98 (p = 0.005) and 7.19 ± 2.74 vs. 3.43 ± 1.64 (p = 0.004) in men and women, respectively. Simple linear regression analysis showed an excellent correlation between epicardial adipose tissue and waist circumference (r = 0.895, p = 0.01) and MRI abdominal VAT (r = 0.864, p = 0.01). Multiple regression analysis showed that epicardial adipose tissue thickness (r² = 0.442, p = 0.02) was the strongest independent variable correlated to MRI VAT. Bland test confirmed the good agreement between the two methods. Discussion: Epicardial adipose tissue showed a strong correlation with anthropometric and imaging measurements of VAT. Hence, transthoracic echocardiography could be an easy and reliable imaging method for VAT prediction.     

Multi-Organ Contribution to the Metabolic Plasma Profile Using Hierarchical Modelling

Hierarchical modelling was applied in order to identify the organs that contribute to the levels of metabolites in plasma. Plasma and organ samples from gut, kidney, liver, muscle and pancreas were obtained from mice. The samples were analysed using gas chromatography time-of-flight mass spectrometry (GC TOF-MS) at the Swedish Metabolomics centre, Umeå University, Sweden. The multivariate analysis was performed by means of principal component analysis (PCA) and orthogonal projections to latent structures (OPLS). The main goal of this study was to investigate how each organ contributes to the metabolic plasma profile. This was performed using hierarchical modelling. Each organ was found to have a unique metabolic profile. The hierarchical modelling showed that the gut, kidney and liver demonstrated the greatest contribution to the metabolic pattern of plasma. For example, we found that metabolites were absorbed in the gut and transported to the plasma. The kidneys excrete branched chain amino acids (BCAAs) and fatty acids are transported in the plasma to the muscles and liver. Lactic acid was also found to be transported from the pancreas to plasma. The results indicated that hierarchical modelling can be utilized to identify the organ contribution of unknown metabolites to the metabolic profile of plasma.     

Prediction of reversible IgG1 aggregation occurring in a size exclusion chromatography column is enabled through a model based approach

One important aspect of antibody separation being studied today is aggregation, as this not only leads to a loss in yield, but aggregates can also be hazardous if injected into the body. The aim of this study was to determine whether the methodology applied in the previous study could be used to predict the aggregation of a different batch of IgG1, and to model the aggregation occurring in a SEC column. Aggregation was found to be reversible. The equilibrium parameter was found to be 272 M^‐1 and the reaction kinetic parameter 1.33 × 10^‐5 s^‐1, both within the 95% confidence interval of the results obtained in the previous work. The effective diffusivities were estimated to be 1.45 × 10^‐13 and 1.90 10^‐14 m²/s for the monomers and dimers, respectively. Good agreement was found between the new model and the chromatograms obtained in the SEC experiments. The model was also able to predict the decrease of dimers due to the dilution and separation in the SEC column during long retention times.     

Selective MicroRNA-Offset RNA Expression in Human Embryonic Stem Cells

Small RNA molecules, including microRNAs (miRNAs), play critical roles in regulating pluripotency, proliferation and differentiation of embryonic stem cells. miRNA-offset RNAs (moRNAs) are similar in length to miRNAs, align to miRNA precursor (pre-miRNA) loci and are therefore believed to derive from processing of the pre-miRNA hairpin sequence. Recent next generation sequencing (NGS) studies have reported the presence of moRNAs in human neurons and cancer cells and in several tissues in mouse, including pluripotent stem cells. In order to gain additional knowledge about human moRNAs and their putative development-related expression, we applied NGS of small RNAs in human embryonic stem cells (hESCs) and fibroblasts. We found that certain moRNA isoforms are notably expressed in hESCs from loci coding for stem cell-selective or cancer-related miRNA clusters. In contrast, we observed only sparse moRNAs in fibroblasts. Consistent with earlier findings, most of the observed moRNAs derived from conserved loci and their expression did not appear to correlate with the expression of the adjacent miRNAs. We provide here the first report of moRNAs in hESCs, and their expression profile in comparison to fibroblasts. Moreover, we expand the repertoire of hESC miRNAs. These findings provide an expansion on the known repertoire of small non-coding RNA contents in hESCs.     

Distinguishing local from global climate influences in the variation of carbon status with altitude in a tree line species

Aim Two alternative hypotheses attempt to explain the upper elevation limit of tree lines world‐wide, the carbon‐limitation hypothesis (CLH) and the growth‐limitation hypothesis (GLH); the altitudinal decrease of temperature is considered the driver constraining either carbon gain or growth. Using a widely distributed tree line species (Nothofagus pumilio) we tested whether tree line altitude is explained by the CLH or the GLH, distinguishing local from global effects. We elaborated expectations based on most probable trends of carbon charging with altitude according to both hypotheses, considering the alternative effects of drought. Location Two climatically contrasting tree line ecotones in the southern Andes of Chile: Mediterranean (36°54′ S) and Patagonia (46°04′ S). Methods At both locations, 35–50 trees of different ages were selected at each of four altitudes (including tree line), and stem and root sapwood tissues were collected to determine non‐structural carbohydrate (NSC) concentrations. NSC accumulates whenever growth is more limited than photosynthesis. An altitudinal increase in NSCs means support for the GLH, while the opposite trend supports the CLH. We also determined stable carbon isotope ratios (δ¹³C) to examine drought constraints on carbon gain. Results NSC concentrations were positively correlated with altitude for stem tissue at the Mediterranean and root sapwood tissue at the Patagonia site. No depletion of NSC was found at either site in either tissue type. For both tissues, mean NSC concentrations were higher for the Patagonia site than for the Mediterranean site. Mean root sapwood NSC concentration values were five times higher than those of the corresponding stem sapwood at all altitudes. Values for δ¹³C were positively correlated with altitude in the Mediterranean site only. Main conclusions We found support for the GLH at the site without drought effects (Patagonia) and no support for the CLH at either site. It is suggested that drought moderated the effects of low temperature by masking the expected trend of the GLH at the Mediterranean site.     

Cloning of the unculturable parasite Pasteuria ramosa and its Daphnia host reveals extreme genotype-genotype interactions

The degree of specificity in host-parasite interactions has important implications for ecology and evolution. Unfortunately, specificity can be difficult to determine when parasites cannot be cultured. In such cases, studies often use isolates of unknown genetic composition, which may lead to an underestimation of specificity. We obtained the first clones of the unculturable bacterium Pasteuria ramosa, a parasite of Daphnia magna. Clonal genotypes of the parasite exhibited much more specific interactions with host genotypes than previous studies using isolates. Clones of P. ramosa infected fewer D. magna genotypes than isolates and host clones were either fully susceptible or fully resistant to the parasite. Our finding enhances our understanding of the evolution of virulence and coevolutionary dynamics in this system. We recommend caution when using P. ramosa isolates as the presence of multiple genotypes may influence the outcome and interpretation of some experiments.