DNA damage, homology-directed repair, and DNA methylation

To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.     

Increasing the volume of vascularized tissue formation in engineered constructs: an experimental study in rats

The authors have previously described a model of in vivo tissue generation based on an implanted, microsurgically created vessel loop in a plastic chamber (volume, 0.45 ml) containing a poly(DL-lactic-co-glycolic acid) (PLGA) scaffold. Tissue grew spontaneously in association with an intense angiogenic sprouting from the loop and almost filled the chamber, resulting in a mean amount of tissue in chambers of 0.23 g with no added matrix scaffold and 0.33 g of tissue in PLGA-filled chambers after 4 weeks of incubation. The aim of the present study was to investigate whether a greater volume of tissue could be generated when the same-size vessel loop was inserted into a larger (1.9 ml) chamber. In four groups of five rats, an arteriovenous shunt sandwiched between two disks of PLGA, used as a scaffold for structural support, was placed inside a large polycarbonate growth chamber. Tissue and PLGA weight and volume, as well as histological characteristics of the newly formed tissue, were assessed at 2, 4, 6, and 8 weeks. Tissue weight and volume showed a strong linear correlation. Tissue weight increased progressively from 0.13 +/- 0.04 g at 2 weeks to 0.57 +/- 0.06 g at 6 weeks (p < 0.0005). PLGA weight decreased progressively from 0.89 +/- 0.07 g at 2 weeks to 0.20 +/- 0.09 g at 8 weeks (p < 0.0005). Histological examination of the specimens confirmed increased tissue growth and maturation over time. It is concluded that larger quantities of tissue can be grown over a longer period of time by using larger-size growth chambers.     

Effect of experience on host preference inCallosobruchus maculatus (Coleoptera: Bruchidae): Variability among populations

Experiments using naive females established differences in host specificity among geographic strains of the seed beetleCallosobruchus maculatus; some females strongly preferred azuki bean over mung bean, and others failed to discriminate between the two hosts. We examined whether such congenital differences affect the degree to which host preference can be modified by experience. In choice tests, previous exposure to azuki bean increased the proportion of eggs laid on that host, but only in strains with a relatively low host specificity. Under more realistic, no-choice conditions, egg-laying experience affected oviposition rates mostly in strains with a high host specificity, but these experiments could not distinguish between the effects of a female's experience per se and her physiological state (i.e., egg load). Our results indicate that the likelihood of detecting an effect of experience on host choice depends on both the experimental protocol and the source of the test population. In natural populations ofC. maculatus, recent egg-laying experience probably plays little or no role in discrimination between host species but may influence discrimination between conspecific seeds that differ in quality.     

Sam68 regulates translation of target mRNAs in male germ cells,necessary for mouse spermatogenesis

Sam68 is a KH-type RNA-binding protein involved in several steps of RNA metabolism with potential implications in cell differentiation and cancer. However, its physiological roles are still poorly understood. Herein, we show that Sam68^−/− male mice are infertile and display several defects in spermatogenesis, demonstrating an essential role for Sam68 in male fertility. Sam68^−/− mice produce few spermatozoa, which display dramatic motility defects and are unable to fertilize eggs. Expression of a subset of messenger mRNAs (mRNAs) is affected in the testis of knockout mice. Interestingly, Sam68 is associated with polyadenylated mRNAs in the cytoplasm during the meiotic divisions and in round spermatids, when it interacts with the translational machinery. We show that Sam68 is required for polysomal recruitment of specific mRNAs and for accumulation of the corresponding proteins in germ cells and in a heterologous system. These observations demonstrate a novel role for Sam68 in mRNA translation and highlight its essential requirement for the development of a functional male gamete.     

Injury-induced insulin resistance in adipose tissue

Hyperglycemia and insulin resistance are common findings in critical illness. Patients in the surgical ICU are frequently treated for this 'critical illness diabetes' with intensive insulin therapy, resulting in a substantial reduction in morbidity and mortality. Adipose tissue is an important insulin target tissue, but it is not known whether adipose tissue is affected by critical illness diabetes. In the present study, a rodent model of critical illness diabetes was used to determine whether adipose tissue becomes acutely insulin resistant and how insulin signaling pathways are being affected. There was a reduction in insulin-induced phosphorylation of IR, IRS-1, Akt and GSK-3β. Since insulin resistance occurs rapidly in adipose tissue, but before the insulin resistance in skeletal muscle, it may play a role in the initial development of critical illness diabetes.     

The diversity of terrestrial isopods in the natural reserve "Saline di Trapani e Paceco" (Crustacea, Isopoda, Oniscidea) in northwestern Sicily

Ecosystems comprising coastal lakes and ponds are important areas for preserving biodiversity. The natural reserve "Saline di Trapani e Paceco" is an interesting natural area in Sicily, formed by the remaining strips of land among salt pans near the coastline. From January 2008 to January 2010, pitfall trapping was conducted in five sampling sites inside the study area. The community of terrestrial isopods was assessed using the main diversity indices. Twenty-four species were collected, only one of them endemic to western Sicily: Porcellio siculoccidentalis Viglianisi, Lombardo & Caruso, 1992. Two species are new to Sicily: Armadilloniscus candidus Budde-Lund, 1885 and Armadilloniscus ellipticus (Harger, 1878). This is high species richness for a single reserve in Sicily. The extended sampling period also allowed us to study species phenology. Most of the species exhibited higher activity in spring than in autumn while some species also exhibited lower activity in the summer. The species richness revealed that the study area is in an acceptable conservation status; Shannon and Pielou indices also confirmed a more or less even distribution of individuals belonging to different species.     

Substituted tetraazaacenaphthylenes as potent CRF1 receptor antagonists for the treatment of depression and anxiety

Two isomers of the hexahydro-tetraazaacenaphthylene templates (1 and 2) are presented as novel, potent, and selective corticotropin releasing factor-1 (CRF1) receptor antagonists. In this paper, we report the affinity and SAR of a series of compounds, as well as pharmacokinetic characterization of a chosen set. The anxiolitic activity of a selected example (2ba) in the rat pup vocalization model is also presented.     

Ferritin as a reporter gene for in vivo tracking of stem cells by 1.5-T cardiac MRI in a rat model of myocardial infarction

The methods currently utilized to track stem cells by cardiac MRI are affected by important limitations, and new solutions are needed. We tested human ferritin heavy chain (hFTH) as a reporter gene for in vivo tracking of stem cells by cardiac MRI. Swine cardiac stem/progenitor cells were transduced with a lentiviral vector to overexpress hFTH and cultured to obtain cardiospheres (Cs). Myocardial infarction was induced in rats, and, after 45 min, the animals were subjected to intramyocardial injection of ～200 hFTH-Cs or nontransduced Cs or saline solution in the border zone. By employing clinical standard 1.5-Tesla MRI scanner and a multiecho T2* gradient echo sequence, we localized iron-accumulating tissue only in hearts treated with hFTH-Cs. This signal was detectable at 1 wk after infarction, and its size did not change significantly after 4 wk (6.33 ± 3.05 vs. 4.41 ± 4.38 mm(2)). Cs transduction did not affect their cardioreparative potential, as indicated by the significantly better preserved left ventricular global and regional function and the 36% reduction in infarct size in both groups that received Cs compared with control infarcts. Prussian blue staining confirmed the presence of differentiated, iron-accumulating cells containing mitochondria of porcine origin. Cs-derived cells displayed CD31, α-smooth muscle, and α-sarcomeric actin antigens, indicating that the differentiation into endothelial, smooth muscle and cardiac muscle lineage was not affected by ferritin overexpression. In conclusion, hFTH can be used as a MRI reporter gene to track dividing/differentiating stem cells in the beating heart, while simultaneously monitoring cardiac morpho-functional changes.