Genome-wide Identification of Structure-Forming Repeats as Principal Sites of Fork Collapse upon ATR Inhibition.

DNA polymerase stalling activates the ATR checkpoint kinase, which in turn suppresses fork collapse and breakage. Herein, we describe use of ATR inhibition (ATRi) as a means to identify genomic sites of problematic DNA replication in murine and human cells. Over 500 high-resolution ATR-dependent sites were ascertained using two distinct methods: replication protein A (RPA)-chromatin immunoprecipitation (ChIP) and breaks identified by TdT labeling (BrITL). The genomic feature most strongly associated with ATR dependence was repetitive DNA that exhibited high structure-forming potential. Repeats most reliant on ATR for stability included structure-forming microsatellites, inverted retroelement repeats, and quasi-palindromic AT-rich repeats. Notably, these distinct categories of repeats differed in the structures they formed and their ability to stimulate RPA accumulation and breakage, implying that the causes and character of replication fork collapse under ATR inhibition can vary in a DNA-structure-specific manner. Collectively, these studies identify key sources of endogenous replication stress that rely on ATR for stability.

Alternative splicing redefines landscape of commonly mutated genes in acute myeloid leukemia.

Most genes associated with acute myeloid leukemia (AML) are mutated in less than 10% of patients, suggesting that alternative mechanisms of gene disruption contribute to this disease. Here, we find a set of splicing events that alter the expression of a subset of AML-associated genes independent of known somatic mutations. In particular, aberrant splicing triples the number of patients with reduced functional EZH2 compared with that predicted by somatic mutation alone. In addition, we unexpectedly find that the nonsense-mediated decay factor DHX34 exhibits widespread alternative splicing in sporadic AML, resulting in a premature stop codon that phenocopies the loss-of-function germline mutations observed in familial AML. Together, these results demonstrate that classical mutation analysis underestimates the burden of functional gene disruption in AML and highlight the importance of assessing the contribution of alternative splicing to gene dysregulation in human disease.

Myocardial infarction remodeling that progresses to heart failure: a signaling misunderstanding.

After myocardial infarction, remodeling of the left ventricle involves a wound-healing orchestra involving a variety of cell types. In order for wound healing to be optimal, appropriate communication must occur; these cells all need to come in at the right time, be activated at the right time in the right amount, and know when to exit at the right time. When this occurs, a new homeostasis is obtained within the infarct, such that infarct scar size and quality are sufficient to maintain left ventricular size and shape. The ideal scenario does not always occur in reality. Often, miscommunication can occur between infarct and remote spaces, across the temporal wound-healing spectrum, and across organs. When miscommunication occurs, adverse remodeling can progress to heart failure. This review discusses current knowledge gaps and recent development of the roles of inflammation and the extracellular matrix in myocardial infarction remodeling. In particular, the macrophage is one cell type that provides direct and indirect regulation of both the inflammatory and scar-forming responses. We summarize current research efforts focused on identifying biomarker indicators that reflect the status of each component of the wound-healing process to better predict outcomes.

Bioactive Scaffolds as a Promising Alternative for Enhancing Critical-Size Bone Defect Regeneration in the Craniomaxillofacial Region.

Reconstruction of critical-size bone defects (CSDs) in the craniomaxillofacial (CMF) region remains challenging. Scaffold-based bone-engineered constructs have been proposed as an alternative to the classical treatments made with autografts and allografts. Scaffolds, a key component of engineered constructs, have been traditionally viewed as biologically passive temporary replacements of deficient bone lacking intrinsic cues to promote osteogenesis. Nowadays, scaffolds are functionalized, giving rise to bioactive scaffolds promoting bone regeneration more effectively than conventional counterparts. This review focuses on the three approaches most used to bioactivate scaffolds: (1) conferring microarchitectural designs or surface nanotopography; (2) loading bioactive molecules; and (3) seeding stem cells on scaffolds, providing relevant examples of in vivo (preclinical and clinical) studies where these methods are employed to enhance CSDs healing in the CMF region. From these, adding bioactive molecules (specifically bone morphogenetic proteins or BMPs) to scaffolds has been the most explored to bioactivate scaffolds. Nevertheless, the downsides of grafting BMP-loaded scaffolds in patients have limited its successful translation into clinics. Despite these drawbacks, scaffolds containing safer, cheaper, and more effective bioactive molecules, combined with stem cells and topographical cues, remain a promising alternative for clinical use to treat CSDs in the CMF complex replacing autografts and allografts.

Parkin Deficiency Suppresses Antigen Presentation to Promote Tumor Immune Evasion and Immunotherapy Resistance.

Parkin is an E3 ubiquitin ligase, which plays a key role in the development of Parkinson disease. Parkin defects also occur in numerous cancers, and a growing body of evidence indicates that Parkin functions as a tumor suppressor that impedes a number of cellular processes involved in tumorigenesis. Here, we generated murine and human models that closely mimic the advanced-stage tumors where Parkin deficiencies are found to provide deeper insights into the tumor suppressive functions of Parkin. Loss of Parkin expression led to aggressive tumor growth, which was associated with poor tumor antigen presentation and limited antitumor CD8+ T-cell infiltration and activation. The effect of Parkin deficiency on tumor growth was lost following depletion of CD8+ T cells. In line with previous findings, Parkin deficiency was linked with mitochondria-associated metabolic stress, PTEN degradation, and enhanced Akt activation. Increased Akt signaling led to dysregulation of antigen presentation, and treatment with the Akt inhibitor MK2206-2HCl restored antigen presentation in Parkin-deficient tumors. Analysis of data from patients with clear cell renal cell carcinoma indicated that Parkin expression was downregulated in tumors and that low expression correlated with reduced overall survival. Furthermore, low Parkin expression correlated with reduced patient response to immunotherapy. Overall, these results identify a role for Parkin deficiency in promoting tumor immune evasion that may explain the poor prognosis associated with loss of Parkin across multiple types of cancer. SIGNIFICANCE: Parkin prevents immune evasion by regulating tumor antigen processing and presentation through the PTEN/Akt network, which has important implications for immunotherapy treatments in patients with Parkin-deficient tumors.

A high-fat diet impacts memory and gene expression of the head in mated female Drosophila melanogaster.

Obesity predisposes humans to a range of life-threatening comorbidities, including type 2 diabetes and cardiovascular disease. Obesity also aggravates neural pathologies, such as Alzheimer's disease, but this class of comorbidity is less understood. When Drosophila melanogaster (flies) are exposed to high-fat diet (HFD) by supplementing a standard medium with coconut oil, they adopt an obese phenotype of decreased lifespan, increased triglyceride storage, and hindered climbing ability. The latter development has been previously regarded as a potential indicator of neurological decline in fly models of neurodegenerative disease. Our objective was to establish the obesity phenotype in Drosophila and identify a potential correlation, if any, between obesity and neurological decline through behavioral assays and gene expression microarray. We found that mated female w1118 flies exposed to HFD maintained an obese phenotype throughout adult life starting at 7 days, evidenced by increased triglyceride stores, diminished life span, and impeded climbing ability. While climbing ability worsened cumulatively between 7 and 14 days of exposure to HFD, there was no corresponding alteration in triglyceride content. Microarray analysis of the mated female w1118 fly head revealed HFD-induced changes in expression of genes with functions in memory, metabolism, olfaction, mitosis, cell signaling, and motor function. Meanwhile, an Aversive Phototaxis Suppression assay in mated female flies indicated reduced ability to recall an entrained memory 6 h after training. Overall, our results support the suitability of mated female flies for examining connections between diet-induced obesity and nervous or neurobehavioral pathology, and provide many directions for further investigation.

RNA-Sequencing of Drosophila melanogaster Head Tissue on High-Sugar and High-Fat Diets.

Obesity has been shown to increase risk for cardiovascular disease and type-2 diabetes. In addition, it has been implicated in aggravation of neurological conditions such as Alzheimer's. In the model organism Drosophila melanogaster, a physiological state mimicking diet-induced obesity can be induced by subjecting fruit flies to a solid medium disproportionately higher in sugar than protein, or that has been supplemented with a rich source of saturated fat. These flies can exhibit increased circulating glucose levels, increased triglyceride content, insulin-like peptide resistance, and behavior indicative of neurological decline. We subjected flies to variants of the high-sugar diet, high-fat diet, or normal (control) diet, followed by a total RNA extraction from fly heads of each diet group for the purpose of Poly-A selected RNA-Sequencing. Our objective was to identify the effects of obesogenic diets on transcriptome patterns, how they differed between obesogenic diets, and identify genes that may relate to pathogenesis accompanying an obesity-like state. Gene ontology analysis indicated an overrepresentation of affected genes associated with immunity, metabolism, and hemocyanin in the high-fat diet group, and CHK, cell cycle activity, and DNA binding and transcription in the high-sugar diet group. Our results also indicate differences in the effects of the high-fat diet and high-sugar diet on expression profiles in head tissue of flies, despite the reportedly similar phenotypic impacts of the diets. The impacted genes, and how they may relate to pathogenesis in the Drosophila obesity-like state, warrant further experimental investigation.

Kinetic Analysis of the Exonuclease Activity of the Bacteriophage T4 Mre11-Rad50 Complex.

Bacteriophage T4 encodes orthologs of the proteins Rad50 (gp46) and Mre11 (gp47), which form a heterotetrameric complex (MR) that is responsible for host genome degradation and the processing of DNA ends for recombination-dependent DNA repair. In this chapter, we describe the ensemble methods currently employed by our laboratory to characterize the exonuclease activity of the T4 MR complex. DNA exonucleases play a vital role in maintaining the integrity of DNA through their participation in DNA repair pathways and as proofreaders for DNA polymerases. Methods for quantifying the general features of the exonuclease, and for determining steady-state kinetic parameters (Km, kcat), the polarity of exonuclease activity, and processivity are presented. These methods should be applicable to all DNA exonucleases, and to some extent endonucleases.

Cytocompatibility of direct water synthesized cadmium selenide quantum dots in colo-205 cells.

Cadmium selenide quantum dots (CdSe QDs), inorganic semiconducting nanocrystals, are alluring increased attraction due to their highly refined chemistry, availability, and super tunable optical properties suitable for many applications in different research areas, such as photovoltaics, light-emitting devices, environmental sciences, and nanomedicine. Specifically, they are being widely used in bio-imaging in contrast to organic dyes due to their high brightness and improved photo-stability, and their ability to tune their absorption and emission spectra upon changing the crystal size. The production of CdSe QDs is mostly assisted by trioctylphosphine oxide compound, which acts as solvent or solubilizing agent and renders the QDs soluble in organic compounds (such as toluene, chloroform, and hexane) that are highly toxic. To circumvent the toxicity-related factor in CdSe QDs, we report the synthesis of CdSe QDs capped with thioglycolic acid (TGA) in an aqueous medium, and their biocompatibility in colo-205 cancer cells. In this study, the [Cd2+]/[TGA] ratio was adjusted to 11:1 and the Se concentration (10 and 15 mM) was monitored in order to evaluate its influence on the optical properties and cytocompatibility. QDs resulted to be quite stable in water (after purification) and RPMI cell medium and no precipitation was observed for long contact times, making them appealing for in vitro experiments. The spectroscopy analysis, advanced electron microscopy, and X-ray diffractometry studies indicate that the final products were successfully formed exhibiting an improved optical response. Colo-205 cells being exposed to different concentrations of TGA-capped CdSe QDs for 12, 24, and 48 h with doses ranging from 0.5 to 2.0 mM show high tolerance reaching cell viabilities as high as 93 %. No evidence of cellular apoptotic pathways was observed as pointed out by our Annexin V assays at higher concentrations. Moreover, confocal microscopy analysis conducted to evaluate the intracellular uptake of TGA-CdSe QDs reveal that the TGA-CdSe QDs were uniformly distributed within the cytosolic side of cell membranes. Our results also suggest that under controlled conditions, direct water-soluble TGA-CdSe QDs can be potentially employed for bio-imaging colo-205 cancer cells with minimal adverse effects.

Tyrosine phosphorylation of the orphan receptor ESDN/DCBLD2 serves as a scaffold for the signaling adaptor CrkL.

A quantitative proteomics screen to identify substrates of the Src family of tyrosine kinases (SFKs) whose phosphorylation promotes CrkL-SH2 binding identified the known Crk-associated substrate (Cas) of Src as well as the orphan receptor endothelial and smooth muscle cell-derived neuropilin-like protein (ESDN). Mutagenesis analysis of ESDN's seven intracellular tyrosines in YxxP motifs found several contribute to the binding of ESDN to the SH2 domains of both CrkCT10 regulator of kinase Crk-Like (CrkL) and a representative SFK Fyn. Quantitative mass spectrometry showed that at least three of these (Y565, Y621 and Y750), as well as non-YxxP Y715, are reversibly phosphorylated. SFK activity was shown to be sufficient, but not required for the interaction between ESDN and the CrkL-SH2 domain. Finally, antibody-mediated ESDN clustering induces ESDN tyrosine phosphorylation and CrkL-SH2 binding.