Propensities of Fatty Acid-Modified ASOs: Self-Assembly vs Albumin Binding.

We conducted a biophysical study to investigate the self-assembling and albumin-binding propensities of a series of fatty acid-modified locked nucleic acid (LNA) antisense oligonucleotide (ASO) gapmers specific to the MALAT1 gene. To this end, a series of biophysical techniques were applied using label-free ASOs that were covalently modified with saturated fatty acids (FAs) of varying length, branching, and 5'/3' attachment. Using analytical ultracentrifugation (AUC), we demonstrate that ASOs conjugated with fatty acids longer than C16 exhibit an increasing tendency to form self-assembled vesicular structures.

Investigation of MicroRNA-134 as a Target against Seizures and SUDEP in a Mouse Model of Dravet Syndrome.

Dravet syndrome (DS) is a catastrophic form of pediatric epilepsy mainly caused by noninherited mutations in the gene. DS patients suffer severe and life-threatening focal and generalized seizures which are often refractory to available anti-seizure medication. Antisense oligonucleotides (ASOs) based approaches may offer treatment opportunities in DS.

AntimiR targeting of microRNA-134 reduces seizures in a mouse model of Angelman syndrome.

Angelman syndrome (AS) is a severe neurodevelopmental disorder featuring ataxia, cognitive impairment, and drug-resistant epilepsy. AS is caused by mutations or deletion of the maternal copy of the paternally imprinted gene, with current precision therapy approaches focusing on re-expression of . Certain phenotypes, however, are difficult to rescue beyond early development.

Antagomir-mediated suppression of microRNA-134 reduces kainic acid-induced seizures in immature mice.

MicroRNAs are short non-coding RNAs that negatively regulate protein levels and perform important roles in establishing and maintaining neuronal network function. Previous studies in adult rodents have detected upregulation of microRNA-134 after prolonged seizures (status epilepticus) and demonstrated that silencing microRNA-134 using antisense oligonucleotides, termed antagomirs, has potent and long-lasting seizure-suppressive effects. Here we investigated whether targeting microRNA-134 can reduce or delay acute seizures in the immature brain.

Effects of obesity on insulin: insulin-like growth factor 1 hybrid receptor expression and Akt phosphorylation in conduit and resistance arteries.

Insulin and insulin-like growth factor-1 stimulate specific responses in arteries, which may be disrupted by diet-induced obesity. We examined (1) temporal effects of high-fat diet compared to low-fat diet in mice on insulin receptor, insulin-like growth factor-1 receptor, insulin receptor/insulin-like growth factor-1 receptor hybrid receptor expression and insulin/insulin-like growth factor-1-mediated Akt phosphorylation in aorta; and (2) effects of high-fat diet on insulin and insulin-like growth factor-1-mediated Akt phosphorylation and vascular tone in resistance arteries. Medium-term high-fat diet (5 weeks) decreased insulin-like growth factor-1 receptor expression and increased hybrid expression (~30%) only.

DBA2J db/db mice are susceptible to early albuminuria and glomerulosclerosis that correlate with systemic insulin resistance.

Diabetic nephropathy (DN) is the leading cause of kidney failure in the world. To understand important mechanisms underlying this condition, and to develop new therapies, good animal models are required. In mouse models of type 1 diabetes, the DBA/2J strain has been shown to be more susceptible to develop kidney disease than other common strains.

A framework for the in vitro evaluation of cancer-relevant molecular characteristics and mitogenic potency of insulin analogues.

Epidemiological and laboratory studies raise the possibility of a link between clinically prescribed insulin analogues and increased cancer risk. Accordingly, there is a regulatory mandate for cancer-related pre-clinical safety evaluation during insulin analogue development, but currently, there is no standardized framework for such in vitro evaluation. We tested human insulin; the super-mitogenic insulin, X10 and insulin-like growth factor I, in four cancer cell lines with a range of insulin-like growth factor-I receptor (IGF-IR)/IR (insulin receptor) ratios (HCT 116, HT-29, COLO 205 and MCF7) and related these to IGF-IR and IR expression in 17 human adenocarcinomas.

Surface-expressed insulin receptors as well as IGF-I receptors both contribute to the mitogenic effects of human insulin and its analogues.

There is a medical need for new insulin analogues. Yet, molecular alterations to the insulin molecule can theoretically result in analogues with carcinogenic effects. Preclinical carcinogenicity risk assessment for insulin analogues rests to a large extent on mitogenicity assays in cell lines.

Strontium is a biased agonist of the calcium-sensing receptor in rat medullary thyroid carcinoma 6-23 cells.

The calcium-sensing receptor (CaSR)-specific allosteric modulator cinacalcet has revolutionized the treatment of secondary hyperparathyroidism in patients with chronic kidney disease. However, its application is limited to patients with end-stage renal disease because of hypocalcemic side effects presumably caused by CaSR-mediated calcitonin secretion from thyroid parafollicular C-cells. These hypocalcemic side effects might be dampened by compounds that bias the signaling of CaSR, causing similar therapeutic effects as cinacalcet without stimulating calcitonin secretion.

Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.

For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection).

Silencing of microRNA-155 in mice during acute inflammatory response leads to derepression of c/ebp Beta and down-regulation of G-CSF.

microRNA-155 (miR-155) has been implicated as a central regulator of the immune system, but its function during acute inflammatory responses is still poorly understood. Here we show that exposure of cultured macrophages and mice to lipopolysaccharide (LPS) leads to up-regulation of miR-155 and that the transcription factor c/ebp Beta is a direct target of miR-155. Interestingly, expression profiling of LPS-stimulated macrophages combined with overexpression and silencing of miR-155 in murine macrophages and human monocytic cells uncovered marked changes in the expression of granulocyte colony-stimulating factor (G-CSF), a central regulator of granulopoiesis during inflammatory responses.

The function of BCL9 in Wnt/beta-catenin signaling and colorectal cancer cells.

Background: Most cases of colorectal cancer are initiated by hyperactivation of the Wnt/beta-catenin pathway due to mutations in the APC tumour suppressor, or in beta-catenin itself. A recently discovered component of this pathway is Legless, which is essential for Wnt-induced transcription during Drosophila development. Limited functional information is available for its two mammalian relatives, BCL9 and B9L/BCL9-2: like Legless, these proteins bind to beta-catenin, and RNAi-mediated depletion of B9L/BCL9-2 has revealed that this protein is required for efficient beta-catenin-mediated transcription in mammalian cell lines.

Frequent hypermethylation of DBC1 in malignant lymphoproliferative neoplasms.

Allelic loss at chromosome 9q31-34 is a frequent event in many lymphoproliferative malignancies. Here, we examined DBC1 at 9q33.1 as a potential target in lymphomagenesis.

Genetic and epigenetic alterations of the reduced folate carrier in untreated diffuse large B-cell lymphoma.

The reduced folate carrier (RFC) is a transmembrane protein that mediates cellular uptake of reduced folates and antifolate drugs, including methotrexate (MTX). Acquired alterations of the RFC gene have been associated with resistance to MTX in cancer cell lines and primary osteosarcomas. Here, we examined RFC for mutations and promoter hypermethylation in (i) the inherently MTX-resistant lymphoma cell line (RL); (ii) 30 paired cases of acute lymphoblastic leukemia (ALL) obtained at diagnosis and at relapse after treatment with MTX; and (iii) 25 cases of diffuse large B-cell lymphoma (DLBCL) at diagnosis, none of which had been previously exposed to MTX.

Genetic and epigenetic alterations of the APC gene in malignant melanoma.

High levels of beta-catenin and activating mutations in the beta-catenin gene (CTNNB1) have been demonstrated in malignant melanomas, implicating dysregulated Wnt signalling in the pathogenesis of this malignancy. We systematically examined melanoma cell lines for activating CTNNB1 mutations as well as genetic and epigenetic alterations of the adenomatous polyposis coli gene (APC), another key component of the Wnt signalling transduction pathway. Of 40 cell lines tested, one carried a truncating APC mutation and loss of the corresponding wild-type allele, and one carried a CTNNB1 missense mutation.

Genetic and epigenetic alterations of the blood group ABO gene in oral squamous cell carcinoma.

Loss of histo-blood group A and B antigen expression is a frequent event in oral carcinomas and is associated with decreased activity of glycosyltransferases encoded by the ABO gene. We examined 30 oral squamous cell carcinomas for expression of A and B antigens and glycosyltransferases. We also examined DNA from these tumors for loss of heterozygosity (LOH) at markers surrounding the ABO locus at chromosome 9q34, for loss of specific ABO alleles, and for hypermethylation of the ABO promoters.

Distinct modes of deregulation of the proto-oncogenic Cdc25A phosphatase in human breast cancer cell lines.

The rapid cell cycle arrest in response to DNA damage is mediated by degradation of the Cdc25A phosphatase, a proto-oncogene whose mRNA is frequently overexpressed in human tumours. Here, we study the occurrence and mechanisms of Cdc25A deregulation in human breast cancer cell lines. We demonstrate aberrantly elevated Cdc25A protein abundance and phosphatase activity in eight out of 15 cell lines, in some cases resulting in abrogation of the Cdc25A-mediated checkpoint response to ionizing radiation (IR), and this defect correlated with hypersensitivity to IR.

DNA methylation: an epigenetic pathway to cancer and a promising target for anticancer therapy.

The unique properties of a cancer cell are acquired through a stepwise accumulation of heritable changes in the information content of proto-oncogenes and tumor suppressor genes. While gain, loss, and mutation of genetic information have long been known to contribute to tumorigenesis, it has been increasingly recognized over the past 5 years that 'epigenetic' mechanisms may play an equally important role. The main epigenetic modification of the human genome is methylation of cytosine residues within the context of the CpG dinucleotide.

ATM mutations are associated with inactivation of the ARF-TP53 tumor suppressor pathway in diffuse large B-cell lymphoma.

The ATM serine-threonine kinase plays a central role in the cellular response to DNA damage. Germ-line mutations in the ATM gene cause ataxia-telangiectasia (A-T), a multisystem disorder associated with predisposition to lymphoma and acute leukemia. Moreover, somatic ATM mutations have been identified in T-cell prolymphocytic leukemia, mantle cell lymphoma, and B-cell chronic lymphocytic leukemia.

Profiling DNA methylation by melting analysis.

The idea of modifying DNA with bisulfite has paved the way for a variety of polymerase chain reaction (PCR) methods for accurately mapping 5-methylcytosine at specific genes. Bisulfite selectively deaminates cytosine to uracil under conditions where 5-methylcytosine remains unreacted. Following conventional PCR amplification of bisulfite-treated DNA, original cytosines appear as thymine while 5-methylcytosines appear as cytosine.