Differences in Gene Expression and Cytokine Release Profiles Highlight the Heterogeneity of Distinct Subsets of Adipose Tissue-Derived Stem Cells in the Subcutaneous and Visceral Adipose Tissue in Humans

Differences in the inherent properties of adipose tissue-derived stem cells (ASC) may contribute to the biological specificity of the subcutaneous (Sc) and visceral (V) adipose tissue depots. In this study, three distinct subpopulations of ASC, i.e. ASC_SVF, ASC_Bottom, and ASC_Ceiling, were isolated from Sc and V fat biopsies of non-obese subjects, and their gene expression and functional characteristics were investigated. Genome-wide mRNA expression profiles of ASC_SVF, ASC_Bottom and ASC_Ceiling from Sc fat were significantly different as compared to their homologous subsets of V-ASCs. Furthermore, ASC_SVF, ASC_Ceiling and ASC_Bottom from the same fat depot were also distinct from each other. In this respect, both principal component analysis and hierarchical clusters analysis showed that ASC_Ceiling and ASC_SVF shared a similar pattern of closely related genes, which was highly different when compared to that of ASC_Bottom. However, larger variations in gene expression were found in inter-depot than in intra-depot comparisons. The analysis of connectivity of genes differently expressed in each ASC subset demonstrated that, although there was some overlap, there was also a clear distinction between each Sc-ASC and their corresponding V-ASC subsets, and among ASC_SVF, ASC_Bottom, and ASC_Ceiling of Sc or V fat depots in regard to networks associated with regulation of cell cycle, cell organization and development, inflammation and metabolic responses. Finally, the release of several cytokines and growth factors in the ASC cultured medium also showed both inter- and intra-depot differences. Thus, ASC_Ceiling and ASC_Bottom can be identified as two genetically and functionally heterogeneous ASC populations in addition to the ASC_SVF, with ASC_Bottom showing the highest degree of unmatched gene expression. On the other hand, inter-depot seem to prevail over intra-depot differences in the ASC gene expression assets and network functions, contributing to the high degree of specificity of Sc and V adipose tissue in humans.     

A new self-compatibility haplotype in the sweet cherry 'Kronio', S5', attributable to a pollen-part mutation in the SFB gene

'Kronio' is a Sicilian cultivar of sweet cherry (Prunus avium), nominally with the incompatibility genotype S(5)S(6), that is reported to be naturally self-compatible. In this work the cause of its self-compatibility was investigated. Test selfing confirmed self-compatibility and provided embryos for analysis; PCR with consensus primers designed to amplify S-RNase and SFB alleles showed that the embryos were of two types, S(5)S(5) and S(5)S(6), indicating that S(6) pollen failed, but S(5) succeeded, perhaps because of a mutation in the pollen or stylar component. Stylar RNase analysis indicated active S-RNases for both S(5) and S(6). The S-RNase alleles were cloned and sequenced; and sequences encode functional proteins. Cloning and sequencing of SFB alleles showed that S(6) was normal but S(5) had a premature stop codon upstream of the variable region HVa resulting in a truncated protein. Therefore, the self-compatibility can be attributed to a pollen-part mutation of S(5), designated S(5)', the first reported case of breakdown of self-incompatibility in diploid sweet cherry caused by a natural mutation at the S-locus. The second intron of the S-RNase associated with S(5)' contained a microsatellite smaller than that associated with S(5); primers designed to amplify across this microsatellite effectively distinguished S(5) from S(5)'. Analysis of some other Sicilian cherries with these primers indicated that S(5)' is also present in the Sicilian cultivar 'Maiolina a Rappu', and this proved to be self-compatible.     

Absorption of horseradish peroxidase in Bombyx mori larval midgut

Increasing experimental evidence indicates that ingested proteins can in part reach the haemocoel undegraded, but information on the mechanisms involved in protein transport across the insect gut is very limited, in spite of the implications that this may have on the development of novel delivery strategies of insecticide proteins targeting haemocoelic receptors. Here we contribute to this field of study, by focusing on horseradish peroxidase (HRP) transport through Bombyx mori larval midgut, isolated and perfused in vitro. The protein crossed the intestinal barrier in a time-dependent manner and the influx was linearly related to time between 30 and 90 min of incubation. HRP absorption was strongly affected by temperature and inhibition of cell metabolism: protein influx at 4 degrees C was reduced to 27% of that measured at 25 degrees C and was similarly inhibited by the metabolic inhibitor DNP. Transmission electron microscopy analysis of midgut columnar cells exposed to HRP showed the presence of the protein both in vesicular structures inside the cytoplasm and in the space between two adjacent absorptive cells, indicating the occurrence of both a transcellular and a paracellular permeation route. The analysis of HRP influx as a function of increasing protein concentration in the lumen supported this morphological indication. The J(max) relative to the HRP transcellular transport component was 121+/-24 pmol/cm(2)/h and the K(d) of the passage through the paracellular route was 1.9+/-0.3 microl/cm(2)/h. The paracellular electrical resistance decreased in midguts exposed to HRP, indicating that its passage through this pathway was likely due to an alteration exerted on the junctional complex by the protein itself. The role of the cytoskeleton in HRP transport was investigated by assessing the impact of drugs affecting microtubules and actin filaments.     

Prep1 (pKnox1) Regulates Mouse Embryonic HSC Cycling and Self-Renewal Affecting the Stat1-Sca1 IFN-Dependent Pathway

A hypomorphic Prep1 mutation results in embryonic lethality at late gestation with a pleiotropic embryonic phenotype that includes defects in all hematopoietic lineages. Reduced functionality of the hematopoietic stem cells (HSCs) compartment might be responsible for the hematopoietic phenotype observed at mid-gestation. In this paper we demonstrate that Prep1 regulates the number of HSCs in fetal livers (FLs), their clonogenic potential and their ability to de novo generate the hematopoietic system in ablated hosts. Furthermore, we show that Prep1 controls the self-renewal ability of the FL HSC compartment as demonstrated by serial transplantation experiments. The premature exhaustion of Prep1 mutant HSCs correlates with the reduced quiescent stem cell pool thus suggesting that Prep1 regulates the self-renewal ability by controlling the quiescence/proliferation balance. Finally, we show that in FL HSCs Prep1 absence induces the interferon signaling pathway leading to premature cycling and exhaustion of fetal HSCs.     

LINE-1 Hypomethylation in Blood and Tissue Samples as an Epigenetic Marker for Cancer Risk: A Systematic Review and Meta-Analysis

Objective

A systematic review and a meta-analysis were carried out in order to summarize the current published studies and to evaluate LINE-1 hypomethylation in blood and other tissues as an epigenetic marker for cancer risk.

Methods

A systematic literature search in the Medline database, using PubMed, was conducted for epidemiological studies, published before March 2014. The random-effects model was used to estimate weighted mean differences (MDs) with 95% Confidence Intervals (CIs). Furthermore, subgroup analyses were conducted by sample type (tissue or blood samples), cancer types, and by assays used to measure global DNA methylation levels. The Cochrane software package Review Manager 5.2 was used.

Results

A total of 19 unique articles on 6107 samples (2554 from cancer patients and 3553 control samples) were included in the meta-analysis. LINE-1 methylation levels were significantly lower in cancer patients than in controls (MD: −6.40, 95% CI: −7.71, −5.09; p<0.001). The significant difference in methylation levels was confirmed in tissue samples (MD −7.55; 95% CI: −9.14, −65.95; p<0.001), but not in blood samples (MD: −0.26, 95% CI: −0.69, 0.17; p = 0.23). LINE-1 methylation levels were significantly lower in colorectal and gastric cancer patients than in controls (MD: −8.33; 95% CI: −10.56, −6.10; p<0.001 and MD: −5.75; 95% CI: −7.75, −3.74; p<0.001) whereas, no significant difference was observed for hepatocellular cancer.

Conclusions

The present meta-analysis adds new evidence to the growing literature on the role of LINE-1 hypomethylation in human cancer and demonstrates that LINE-1 methylation levels were significantly lower in cancer patients than in control samples, especially in certain cancer types. This result was confirmed in tissue samples, both fresh/frozen or FFPE specimens, but not in blood. Further studies are needed to better clarify the role of LINE-1 methylation in specific subgroups, considering both cancer and sample type, and the methods of measurement.     

Yeast Frataxin Is Stabilized by Low Salt Concentrations: Cold Denaturation Disentangles Ionic Strength Effects from Specific Interactions

Frataxins are a family of metal binding proteins associated with the human Friedreich''s ataxia disease. Here, we have addressed the effect of non-specifically binding salts on the stability of the yeast ortholog Yfh1. This protein is a sensitive model since its stability is strongly dependent on the environment, in particular on ionic strength. Yfh1 also offers the unique advantage that its cold denaturation can be observed above the freezing point of water, thus allowing the facile construction of the whole protein stability curve and hence the measurement of accurate thermodynamic parameters for unfolding. We systematically measured the effect of several cations and, as a control, of different anions. We show that, while strongly susceptible to ionic strength, as it would be in the cellular environment, Yfh1 stability is sensitive not only to divalent cations, which bind specifically, but also to monovalent cations. We pinpoint the structural bases of the stability and hypothesize that the destabilization induced by an unusual cluster of negatively charged residues favours the entrance of water molecules into the hydrophobic core, consistent with the generally accepted mechanism of cold denaturation.     

Human Hsp60 with Its Mitochondrial Import Signal Occurs in Solution as Heptamers and Tetradecamers Remarkably Stable over a Wide Range of Concentrations

It has been established that Hsp60 can accumulate in the cytosol in various pathological conditions, including cancer and chronic inflammatory diseases. Part or all of the cytosolic Hsp60 could be naïve, namely, bear the mitochondrial import signal (MIS), but neither the structure nor the in solution oligomeric organization of this cytosolic molecule has still been elucidated. Here we present a detailed study of the structure and self-organization of naïve cytosolic Hsp60 in solution. Results were obtained by different biophysical methods (light and X ray scattering, single molecule spectroscopy and hydrodynamics) that all together allowed us to assay a wide range of concentrations of Hsp60. We found that Naïve Hsp60 in aqueous solution is assembled in very stable heptamers and tetradecamers at all concentrations assayed, without any trace of monomer presence.     

Reversible acetylation regulates vascular endothelial growth factor receptor-2 activity

The tyrosine kinase receptor vascular endothelial growth factor receptor 2 （VEG FR2） is a key regulator of angiogenesis. Here we show that VEGFR2 is acetylated in endothelial cells both at four lysine residues forming a dense cluster in the kinase insert domain and at a single lysine located in the receptor activation loop. These modifications are under dynamic control of the acetyltransferase p300 and two deacetyiases HDAC5 and HDAC6. We demonstrate that VEGFR2 acetylation essentially regulates receptor phosphorylation. In par- ticular, VEGFR2 acetylation significantly alters the kinetics of receptor phosphorylation after ligand binding, allowing receptor phos- phoryiation and intraceUular signaling upon proLonged stimulation with VEGF. Molecular dynamics simulations indicate that acetylation of the lysine in the activation loop contributes to the transition to an open active state, in which tyrosine phosphorylation is favored by better exposure of the kinase target residues. These findings indicate that post-translational modification by acetyiation is a critical mechanism that directLy affects VEGFR2 function.