Tcl1 protein functions as an inhibitor of de novo DNA methylation in B-cell chronic lymphocytic leukemia (CLL)

B-cell chronic lymphocytic leukemia (CLL) is the most common human leukemia. Deregulation of the T-cell leukemia/lymphoma 1 oncogene (TCL1) in mouse B cells causes a CD5(+) leukemia similar to aggressive human CLL. To examine the mechanisms by which Tcl1 protein exerts its oncogenic activity in B cells, we performed proteomics experiments to identify its interacting partners. We found that Tcl1 physically interacts with de novo DNA methylthansferases Dnmt3A and Dnmt3B. We further investigated the effects of Tcl1 up-regulation on the enzymatic activity of Dnmt3A and found that Tcl1 overexpression drastically inhibits Dnmt3A function. In addition, B cells from TCL1 transgenic mice showed a significant decrease in DNA methylation compared with WT controls. Similarly, CLL samples with high Tcl1 expression showed a decrease in DNA methylation compared with CLL samples with low Tcl1 expression. Given the previous reports of inactivating mutations of DNMT3A in acute myelogenous leukemia and myelodysplastic syndrome, our results suggest that inhibition of de novo DNA methylation may be a common oncogenic mechanism in leukemogenesis.     

Thieving rodents as substitute dispersers of megafaunal seeds

The Neotropics have many plant species that seem to be adapted for seed dispersal by megafauna that went extinct in the late Pleistocene. Given the crucial importance of seed dispersal for plant persistence, it remains a mystery how these plants have survived more than 10,000 y without their mutualist dispersers. Here we present support for the hypothesis that secondary seed dispersal by scatter-hoarding rodents has facilitated the persistence of these large-seeded species. We used miniature radio transmitters to track the dispersal of reputedly megafaunal seeds by Central American agoutis, which scatter-hoard seeds in shallow caches in the soil throughout the forest. We found that seeds were initially cached at mostly short distances and then quickly dug up again. However, rather than eating the recovered seeds, agoutis continued to move and recache the seeds, up to 36 times. Agoutis dispersed an estimated 35% of seeds for >100 m. An estimated 14% of the cached seeds survived to the next year, when a new fruit crop became available to the rodents. Serial video-monitoring of cached seeds revealed that the stepwise dispersal was caused by agoutis repeatedly stealing and recaching each other's buried seeds. Although previous studies suggest that rodents are poor dispersers, we demonstrate that communities of rodents can in fact provide highly effective long-distance seed dispersal. Our findings suggest that thieving scatter-hoarding rodents could substitute for extinct megafaunal seed dispersers of tropical large-seeded trees.     

In situ observation of peptide bond formation at the water–air interface

We report unambiguous spectroscopic evidence of peptide bond formation at the air–water interface, yielding a possible mechanism providing insight into the formation of modern ribosomal peptide bonds, and a means for the emergence of peptides on early Earth. Protein synthesis in aqueous environments, facilitated by sequential amino acid condensation forming peptides, is a ubiquitous process in modern biology, and a fundamental reaction necessary in prebiotic chemistry. Such reactions, however, are condensation reactions, requiring the elimination of a water molecule for every peptide bond formed, and are thus unfavorable in aqueous environments both from a thermodynamic and kinetic point of view. We use the hydrophobic environment of the air–water interface as a favorable venue for peptide bond synthesis, and demonstrate the occurrence of this chemistry with in situ techniques using Langmuir-trough methods and infrared reflection absorption spectroscopy. Leucine ethyl ester (a small amino acid ester) first partitions to the water surface, then coordinates with Cu²⁺ ions at the interface, and subsequently undergoes a condensation reaction selectively forming peptide bonds at the air–water interface.Protein synthesis (condensation of amino acids through sequential peptide bond formation) is a fundamental and ubiquitous reaction in biology. Aqueous media are the required environments in which this chemistry takes place; however, protein synthesis is unfavorable in aqueous solution. In modern biology, the condensation reactions necessary in the formation of peptide bonds are facilitated catalytically by the large subunit of the ribosome. The mechanism of this catalyzed reaction originally proposed by Nissen et al. in 2000 (1) involved favorable orientation of peptide precursors, acid-base catalysis and transition-state stabilization, and the altered pK_a of the functional groups of the precursors caused by the reaction environment provided by the active site; such pK_a shifts had previously been seen in the active sites of other proteins (2). Since then, the original mechanism has been contested (3, 4). The acknowledged mechanistic function of the ribosome’s active site is its ability to bring the precursors in close proximity and orient them for reaction, with further mechanistic details remaining unresolved (4). Studies of peptide bond formation in the absence of modern biological machinery can give insight into the mechanism employed by the ribosome’s active site, as well as yield important information in the prebiotic route to the first peptides in the origin of life. The formation of a peptide bond (reaction R1 shown below) is a condensation reaction, eliminating a water molecule for each peptide bond formed, and thus faces both thermodynamic and kinetic constraints in bulk aqueous solution (5).5, 6). Amino acid monomers have the added kinetic disadvantage of existing primarily as zwitterions at environmentally and physiologically relevant pH values in bulk aqueous solution (5, 7). Insightful experiments have been performed, yielding peptide bonds in anhydrous solvents with amino acid ester starting materials and copper(II) ion catalysis (8, 9). Transition metal ions are thought to have been components of the early ocean (10), with one source being the heavy meteoritic and cometary bombardment experienced by the early Earth, but the anhydrous solvents used in these studies are neither physiologically relevant nor likely to have been present on early Earth. The same mechanism was attempted in aqueous solution, but no peptide formation was detected (9). Polymer formation in aqueous environments would most likely have been necessary on early Earth because the liquid ocean would have been the reservoir of amino acid precursors needed for protein synthesis. In this work, the air–water interface is utilized as the auspicious environment for peptide bond formation, coupling the water surface with the bulk water reservoir of monomers. In situ surface-sensitive techniques are used here to observe the condensation reaction of a model system composed of a small, water-soluble amino acid ester (leucine ethyl ester) through Cu²⁺ coordination.Air–water interfaces are found now, as on prebiotic Earth, at the surfaces of lakes, oceans, and atmospheric aerosols. The air–water interface (atmospheric aerosols in particular) has previously been proposed to be important in prebiotic chemistry (11–14) because it provides a unique environment for chemistry through its ability to concentrate and align biochemical precursors and to alter the state of ionization of surface species (15–19). Contemporary marine aerosols have been found to contain the amino acid precursors necessary for peptide bond chemistry (20), enabling the possibility for their use in such reactions. Further, the fluctuating conditions experienced by aerosols throughout their atmospheric lifetime, including evaporation of water, coagulation, and possibly reentry into the ocean, would naturally provide the compression of the surface layer shown in this work to be necessary for Cu²⁺ coordination leading to peptide bond chemistry (12).In addition, the unfavorable equilibrium constant for peptide bond formation in bulk aqueous solution is shifted when the molecules experience a water-restricted reaction environment at the water surface. Although the exact surface pH of water is debated (21–23), the surface is known to alter the pK_a of surface-active molecules toward their neutral form (24, 25), which aids in the promotion of peptide bond chemistry at the interface by reducing zwitterion formation and alleviating the kinetic constraint on peptide bond synthesis. The air–water interface has been reported in the literature to have a catalytic role in peptide bond formation (26–29) using synthetic long-chain amino acid esters that are anchored to the surface by the polar groups attached to their 18-carbon-long hydrocarbon tails, thus forced to reside solely at the surface of the water. Reaction amongst the surface monomers was promoted in these studies (27–29) through surface compression, and supported by subsequent collection, drying, and analysis of the surface products. In the work presented here, infrared reflection absorption spectroscopy (IRRAS) and Langmuir trough methods were used to observe, in situ, complex formation with a metal cation followed by condensation chemistry leading to peptide bond formation occurring at the air–water interface. The observation of such condensation reactions in situ at the interface and with such a small activation group (an ethyl ester) on the starting amino acid precursor in an aqueous environment is unique.     

Addition of iron to erythropoiesis-stimulating agents in cancer patients: a meta-analysis of randomized trials

Background  

Iron supplementation could improve the hematopoietic response of erythropoiesis-stimulating agents (ESAs) used for chemotherapy-induced anemia.     

L-citrulline Prevents Alveolar and Vascular Derangement in a Rat Model of Moderate Hyperoxia-induced Lung Injury

Background

Moderate normobaric hyperoxia causes alveolar and vascular lung derangement in the newborn rat. Endogenous nitric oxide (NO), which promotes lung growth, is produced from the metabolism of l-arginine to l-citrulline in endothelial cells. We investigated whether administering l-citrulline by raising the serum levels of l-arginine and enhancing NO endogenous synthesis attenuates moderate hyperoxia-induced lung injury.

Methods

Newborn rats were exposed to FiO₂?=?0.6 or room air for 14?days to induce lung derangement and then were administered l-citrulline or a vehicle (sham). Lung histopathology was studied with morphometric features. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected for analysis. Lung vascular endothelial growth factor (VEGF), nitric oxide synthase (eNOS), and matrix metalloproteinase 2 (MMP2) gene and protein expressions were assessed.

Results

Serum l-arginine rose in the L-citr?+?hyperoxia group (p?=?0.05), as well as the Von Willebrand factor stained vessels count (p?=?0.0008). Lung VEGF immune staining, localized on endothelial cells, was weaker in the sections under hyperoxia than the l-citr?+?hyperoxia and room air groups. This pattern was comparable with the VEGF gene and protein expression profiles. Mean alveolar size increased in the untreated hyperoxia and sham-treated groups compared with the groups reared in room air or treated with l-citrulline under exposure to hyperoxia (p?=?0.0001). Lung VEGF and eNOS increased in the l-citrulline-treated rats, though this treatment did not change MMP2 gene expression but regulated the MMP2 active protein, which rose in BALF (p?=?0.003).

Conclusions

We conclude that administering l-citrulline proved effective in improving alveolar and vascular growth in a model of oxygen-induced pulmonary damage, suggesting better lung growth and matrix regulation than in untreated groups.     

Identification of a Prognostic Signature Based on the Expression of Genes Related to the Insulin Pathway in Early Breast Cancer

IntroductionInsulin and the insulin-like growth factor (IGF) family play a key role in breast cancer (BC).ObjectiveIn this study, we evaluated on a genomic scale the potential prognostic value of insulin signaling in early BC.MethodsCandidate genes were selected from the published literature and gene expression profiling experiments. Three publicly available BC datasets, containing gene expression data on 502 cases, were used to test the prognostic ability of the score. The gene signature was developed on {"type":"entrez-geo","attrs":{"text":"GSE1456","term_id":"1456"}}GSE1456, containing microarray data from 159 patients, split into a training set (102 breast tumors) and a validation set (n = 57). {"type":"entrez-geo","attrs":{"text":"GSE3494","term_id":"3494"}}GSE3494 and {"type":"entrez-geo","attrs":{"text":"GSE2990","term_id":"2990"}}GSE2990 (350 patients) were used for external validation. Univariate Mann-Whitney test was used to identify genes differentially expressed between relapsed and nonrelapsed patients. Expression of genes significantly correlated with relapse was combined in a linear score. Patients were classified as low or high risk with respect to the median value.ResultsOn the training set, 15 genes turned out to be differentially expressed: 8-year disease-free survival (DFS) was 51 and 91% in the high- and low-risk group (p < 0.001), respectively. In the validation set, DFS was 97 and 54% (p = 0.009), respectively. External validation: 8-year DFS was 72 and 61%, respectively, in {"type":"entrez-geo","attrs":{"text":"GSE3494","term_id":"3494"}}GSE3494 (p = 0.03) and 74 and 55% in {"type":"entrez-geo","attrs":{"text":"GSE2990","term_id":"2990"}}GSE2990 (p = 0.03). By multivariate analyses, the insulin signature was significantly associated with DFS, independently of age, hormone receptor status, nodal status, and grade.ConclusionsOur findings indicate that the insulin pathway is involved in BC prognosis at a genomic level and provide a window of selectivity for preventive and treatment strategies targeting the insulin/IGF pathway in BC patients.     

Shear bond strength evaluation of bonded molar tubes on fluorotic molars

Objective:To study the shear bond strength (SBS), sites of failure, and micromorphology of bonded molar tubes used on teeth affected by dental fluorosis.Materials and Methods:This in vitro study included 140 first molars classified according to Dean''s index for dental fluorosis. Samples were divided into seven groups: (1) healthy teeth etched for 15 seconds, (2) teeth with moderate fluorosis (MOF) etched for 15 seconds, (3) teeth with MOF etched for 150 seconds, (4) teeth with MOF microabrasion etched for 15 seconds, (5) teeth with severe fluorosis (SEF) etched for 15 seconds, (6) teeth with SEF etched for 150 seconds, and (7) teeth with SEF microabrasion etched for 15 seconds. All samples were incubated and were then submitted to the SBS test and evaluated with the modified adhesive remnant index (ARI) and analyzed by using a scanning electronic microscope.Results:The SBS mean value for healthy enamel was 20 ± 10.2 MPa. For the group with MOF, the etched 150-second mean value was the highest (19 ± 7.6 MPa); for the group with SEF treated with microabrasion and etched for 15 seconds, the mean value was (13 ± 4.1 MPa). Significant differences (P ≤ .05) were found in the ARI between healthy and fluorosed groups.Conclusions:Fluorotic enamel affects the adhesion of bonded molar tubes. The use of overetching in cases of MOF and the combination of microabrasion and etching in SEF provides a suitable adhesion for fixed appliance therapy.