Interaction of oligonucleotide-based amphiphilic block copolymers with cell membrane models

Oligonucleotides have unique molecular recognition properties, being involved in biological mechanisms such as cell-surface receptor recognition or gene silencing. For their use in human therapy for drug or gene delivery, the cell membrane remains a barrier, but this can be obviated by grafting a hydrophobic tail to the oligonucleotide. Here we demonstrate that two oligonucleotides, one consisting of 12 guanosine units (G(12)), and the other one consisting of five adenosine and seven guanosine (A(5)G(7)) units, when functionalized with poly(butadiene), namely PB-G(12) and PB-A(5)G(7), can be inserted into Langmuir monolayers of dipalmitoyl phosphatidyl choline (DPPC), which served as a cell membrane model. PB-G(12) and PB-A(5)G(7) were found to affect the DPPC monolayer even at high surface pressures. The effects from PB-G(12) were consistently stronger, particularly in reducing the elasticity of the DPPC monolayers, which may have important biological implications. Multilayers of DPPC and nucleotide-based copolymers could be adsorbed onto solid supports, in the form of Y-type LB films, in which the molecular-level interaction led to lower energies in the vibrational spectra of the nucleotide-based copolymers. This successful deposition of solid films opens the way for devices to be produced which exploit the molecular recognition properties of the nucleotides. (C) 2010 Elsevier Inc. All rights reserved.

Interaction of cationic bilayer fragments with a model oligonucleotide

The interaction between cationic bilayer fragments and a model oligonucleotide was investigated by differential scanning calorimetry, turbidimetry, determination of excimer to monomer ratio of 2-(10-(1-pyrene)-decanoyl)-phosphatidyl-choline in bilayer fragment dispersions and dynamic light scattering for sizing and zeta-potential analysis. Salt (Na(2)HPO(4)), mononucleotide (2`-deoxyadenosine-5`-monophosphate) or poly (dA) oligonucleotide (3`-AAA AAA AAA A-5`) affected structure and stability of dioctadecyldimethylammonium bromide bilayer fragments. Oligonucleotide and salt increased bilayer packing due to bilayer fragment fusion. Mononucleotide did not reduce colloid stability or did not cause bilayer fragment fusion. Charge neutralization of bilayer fragments by poly (dA) at 1:10 poly (dA):dioctadecyldimethylammonium bromide molar ratio caused extensive aggregation, maximal size and zero of zeta-potential for the assemblies. Above charge neutralization, assemblies recovered colloid stability due to charge overcompensation. For bilayer fragments/poly (dA), the nonmonotonic behavior of colloid stability as a function of poly (dA) concentration was unique for the oligonucleotide and was not observed for Na(2)HPO(4) or 2`-deoxyadenosine-5`-monophosphate. For the first time, such interactions between cationic bilayer fragments and mono- or oligonucleotide were described in the literature. Bilayer fragments/oligonucleotide assemblies may find interesting applications in drug delivery. (c) 2010 Elsevier B.V. All rights reserved.

Consensus Statement: Chromosomal Microarray Is a First-Tier Clinical Diagnostic Test for Individuals with Developmental Disabilities or Congenital Anomalies

Chromosomal microarray (CMA) is increasingly utilized for genetic testing of individuals with unexplained developmental delay/intellectual disability (DD/ID), autism spectrum disorders (ASD), or multiple congenital anomalies (MCA). Performing CMA and G-banded karyotyping on every patient substantially increases the total cost of genetic testing. The International Standard Cytogenomic Array (ISCA) Consortium held two international workshops and conducted a literature review of 33 studies, including 21,698 patients tested by CMA. We provide an evidence-based summary of clinical cytogenetic testing comparing CMA to G-banded karyotyping with respect to technical advantages and limitations, diagnostic yield for various types of chromosomal aberrations, and issues that affect test interpretation. CMA offers a much higher diagnostic yield (15%-20%) for genetic testing of individuals with unexplained DD/ID, ASD, or MCA than a G-banded karyotype (similar to 3%, excluding Down syndrome and other recognizable chromosomal syndromes), primarily because of its higher sensitivity for submicroscopic deletions and duplications. Truly balanced rearrangements and low-level mosaicism are generally not detectable by arrays, but these are relatively infrequent causes of abnormal phenotypes in this population (<1%). Available evidence strongly supports the use of CMA in place of G-banded karyotyping as the first-tier cytogenetic diagnostic test for patients with DD/ID, ASD, or MCA. G-banded karyotype analysis should be reserved for patients with obvious chromosomal syndromes (e.g., Down syndrome), a family history of chromosomal rearrangement, or a history of multiple miscarriages.

Characterization of microsatellite loci of Tetragonisca angustula (Hymenoptera, Apidae, Meliponini)

An enriched genomic library was constructed from Tetragonisca angustula, a stingless bee species widely distributed in Brazil. The library was screened using two simple-repeat oligonucleotide probes and 21 microsatellite primer pairs were designed flanking a selection of repeat sequences within positive clones. The polymorphism of the microsatellite loci was analyzed by screening a sample of 19 unrelated T. angustula workers. Fifteen out of 21 loci were shown to be polymorphic, with observed heterozygosity estimates ranging from 0.00 to 0.89. The primers were also successfully used to amplify microsatellite loci from other stingless bee species, Tetragonisca fiebrigi, Tetragonisca weyrauchi, Lestrimelitta maracaia and Schwarziana quadripunctata. The results from variability analyses suggest that the microsatellite loci isolated from T. angustula will be useful in further population studies for the species and also for other Meliponini.

Complex rearrangements in patients with duplications of MECP2 can occur by fork stalling and template switching

Duplication at the Xq28 band including the MECP2 gene is one of the most common genomic rearrangements identified in neurodevelopmentally delayed males. Such duplications are non-recurrent and can be generated by a non-homologous end joining (NHEJ) mechanism. We investigated the potential mechanisms for MECP2 duplication and examined whether genomic architectural features may play a role in their origin using a custom designed 4-Mb tiling-path oligonucleotide array CGH assay. Each of the 30 patients analyzed showed a unique duplication varying in size from similar to 250 kb to similar to 2.6 Mb. Interestingly, in 77% of these non-recurrent duplications, the distal breakpoints grouped within a 215 kb genomic interval, located 47 kb telomeric to the MECP2 gene. The genomic architecture of this region contains both direct and inverted low-copy repeat (LCR) sequences; this same region undergoes polymorphic structural variation in the general population. Array CGH revealed complex rearrangements in eight patients; in six patients the duplication contained an embedded triplicated segment, and in the other two, stretches of non-duplicated sequences occurred within the duplicated region. Breakpoint junction sequencing was achieved in four duplications and identified an inversion in one patient, demonstrating further complexity. We propose that the presence of LCRs in the vicinity of the MECP2 gene may generate an unstable DNA structure that can induce DNA strand lesions, such as a collapsed fork, and facilitate a Fork Stalling and Template Switching event producing the complex rearrangements involving MECP2.

Clinical and molecular delineation of the 17q21.31 microdeletion syndrome

Background: The chromosome 17q21.31 microdeletion syndrome is a novel genomic disorder that has originally been identified using high resolution genome analyses in patients with unexplained mental retardation. Aim: We report the molecular and/or clinical characterisation of 22 individuals with the 17q21.31 microdeletion syndrome. Results: We estimate the prevalence of the syndrome to be 1 in 16 000 and show that it is highly underdiagnosed. Extensive clinical examination reveals that developmental delay, hypotonia, facial dysmorphisms including a long face, a tubular or pear-shaped nose and a bulbous nasal tip, and a friendly/amiable behaviour are the most characteristic features. Other clinically important features include epilepsy, heart defects and kidney/urologic anomalies. Using high resolution oligonucleotide arrays we narrow the 17q21.31 critical region to a 424 kb genomic segment (chr17: 41046729-41470954, hg17) encompassing at least six genes, among which is the gene encoding microtubule associated protein tau (MAPT). Mutation screening of MAPT in 122 individuals with a phenotype suggestive of 17q21.31 deletion carriers, but who do not carry the recurrent deletion, failed to identify any disease associated variants. In five deletion carriers we identify a <500 bp rearrangement hotspot at the proximal breakpoint contained within an L2 LINE motif and show that in every case examined the parent originating the deletion carries a common 900 kb 17q21.31 inversion polymorphism, indicating that this inversion is a necessary factor for deletion to occur (p< 10(25)). Conclusion: Our data establish the 17q21.31 microdeletion syndrome as a clinically and molecularly well recognisable genomic disorder.

Association of NAD(P)H Oxidase with Glucose-Induced Insulin Secretion by Pancreatic beta-Cells

We previously described the presence of nicotinamide adenine dinucleotide phosphate reduced form [NAD(P)H] oxidase components in pancreatic beta-cells and its activation by glucose, palmitic acid, and proinflammatory cytokines. In the present study, the importance of the NAD(P)H oxidase complex for pancreatic beta-cell function was examined. Rat pancreatic islets were incubated in the presence of glucose plus diphenyleneiodonium, a NAD(P)H oxidase inhibitor, for 1 h or with the antisense oligonucleotide for p47(PHOX) during 24 h. Reactive oxygen species (ROS) production was determined by a fluorescence assay using 2,7-dichlorodihydrofluorescein diacetate. Insulin secretion, intracellular calcium responses, [U-(14)C] glucose oxidation, and expression of glucose transporter-2, glucokinase and insulin genes were examined. Antisense oligonucleotide reduced p47(PHOX) expression [an important NAD(P)H oxidase cytosolic subunit] and similarly to diphenyleneiodonium also blunted the enzyme activity as indicated by reduction of ROS production. Suppression of NAD(P)H oxidase activity had an inhibitory effect on intracellular calcium responses to glucose and glucose-stimulated insulin secretion by isolated islets. NAD(P)H oxidase inhibition also reduced glucose oxidation and gene expression of glucose transporter-2 and glucokinase. These findings indicate that NAD(P)H oxidase activation plays an important role for ROS production by pancreatic beta-cells during glucose-stimulated insulin secretion. The importance of this enzyme complex for the beta-cell metabolism and the machinery involved in insulin secretion were also shown. (Endocrinology 150: 2197-2201, 2009)

The role of proliferator-activated receptor gamma coactivator-1 alpha in the fatty-acid -dependent transcriptional control of interleukin-10 in hepatic cells of rodents

Interleukin-10 (IL-10) is an endogenous factor that restrains hepatic insulin resistance in diet-induced steatosis Reducing IL-10 expression increases proinflammatory activity in the steatotic liver and worsens insulin resistance As the transcriptional coactivator proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) plays a central role in dysfunctional hepatocytic activity in diet-induced steatosis, we hypothesized that at least part of the action of PGC-1 alpha could be mediated by reducing the transcription of the IL-10 gene Here, we used immunoblotting, real-time polymerase chain reaction, immunocytochemistry, and chromatin immunoprecipitation assay to investigate the role of PGC-1 alpha in the control of IL-10 expression in hepatic cells First, we show that, in the intact steatotic liver, the expressions of IL-10 and PGC-1 alpha are increased Inhibiting PGC-1 alpha expression by antisense oligonucleotide increases IL-10 expression and reduces the steatotic phenotype. In cultured hepatocytes, the treatment with saturated and unsaturated fatty acids increased IL-10 expression. This was accompanied by increased association of PGC-1 alpha with c-Maf and p50-nuclear factor (NF) kappa B, 2 transcription factors known to modulate IL-10 expression In addition, after fatty acid treatment. PGC-1 alpha, c-Maf, and p50-NF kappa B migrate from the cytosol to the nuclei of hepatocytes and bind to the IL-10 promoter region Inhibiting NF kappa B activation with salicylate reduces IL-10 expression and the association of PGC-1 alpha with p50-NF kappa B Thus, PGC-1 alpha emerges as a potential transcriptional regulator of the inflammatory phenomenon taking place in the steatotic liver (C) 2010 Elsevier Inc All rights reserved

Inflammation of the Hypothalamus Leads to Defective Pancreatic Islet Function

Type 2 diabetes mellitus results from the complex association of insulin resistance and pancreatic beta-cell failure. Obesity is the main risk factor for type 2 diabetes mellitus, and recent studies have shown that, in diet-induced obesity, the hypothalamus becomes inflamed and dysfunctional, resulting in the loss of the perfect coupling between caloric intake and energy expenditure. Because pancreatic beta-cell function is, in part, under the control of the autonomic nervous system, we evaluated the role of hypothalamic inflammation in pancreatic islet function. In diet-induced obesity, the earliest markers of hypothalamic inflammation are present at 8 weeks after the beginning of the high fat diet; similarly, the loss of the first phase of insulin secretion is detected at the same time point and is restored following sympathectomy. Intracerebroventricular injection of a low dose of tumor necrosis factor a leads to a dysfunctional increase in insulin secretion and activates the expression of a number of markers of apoptosis in pancreatic islets. In addition, the injection of stearic acid intracerebroventricularly, which leads to hypothalamic inflammation through the activation of tau-like receptor-4 and endoplasmic reticulum stress, produces an impairment of insulin secretion, accompanied by increased expression of markers of apoptosis. The defective insulin secretion, in this case, is partially dependent on sympathetic signal-induced peroxisome proliferator receptor-gamma coactivator Delta a and uncoupling protein-2 expression and is restored after sympathectomy or following PGC1 alpha expression inhibition by an antisense oligonucleotide. Thus, the autonomic signals generated in concert with hypothalamic inflammation can impair pancreatic islet function, a phenomenon that may explain the early link between obesity and defective insulin secretion.

Brainstem oxytocinergic modulation of heart rate control in rats: effects of hypertension and exercise training

Oxytocinergic brainstem projections participate in the autonomic control of the circulation. We investigated the effects of hypertension and training on cardiovascular parameters after oxytocin (OT) receptor blockade within the nucleus tractus solitarii (NTS) and NTS OT and OT receptor expression. Male spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were trained (55% of maximal exercise capacity) or kept sedentary for 3 months and chronically instrumented (NTS and arterial cannulae). Mean arterial blood pressure (MAP) and heart rate (HR) were measured at rest and during an acute bout of exercise after NTS pretreatment with vehicle or OT antagonist (20 pmol of OT antagonist (200 nl of vehicle)-1). Oxytocin and OT receptor were quantified (35S-oligonucleotide probes, in situ hybridization) in other groups of rats. The SHR exhibited high MAP and HR (P < 0.05). Exercise training improved treadmill performance and reduced basal HR (on average -11%) in both groups, but did not change basal MAP. Blockade of NTS OT receptor increased exercise tachycardia only in trained groups, with a larger effect on trained WKY rats (+31 +/- 9 versus +12 +/- 3 beats min-1 in the trained SHR). Hypertension specifically reduced NTS OT receptor mRNA density (-46% versus sedentary WKY rats, P < 0.05); training did not change OT receptor density, but significantly increased OT mRNA expression (+2.5-fold in trained WKY rats and +15% in trained SHR). Concurrent hypertension- and training-induced plastic (peptide/receptor changes) and functional adjustments (HR changes) of oxytocinergic control support both the elevated basal HR in the SHR group and the slowing of the heart rate (rest and exercise) observed in trained WKY rats and SHR.

Protein Assembly onto Cationic Supported Bilayers

Cationic supported bilayers on latex are useful to isolate and immobilize oppositely charged proteins as a monomolecular layer over a range of low protein concentrations and particle number densities. Cholera toxin (CT) from Vibrio cholerae, an 87 kDa AB(5) hexameric protein and bovine serum albumin (BSA) self-assembled on dioctadecyldimethylammonium bromide (DODAB) supported bilayers with high affinity yielding highly organized and monodisperse particulates at 5 x 10(9) particles/mL, over a range of low protein concentrations (0-0.025 mg/mL BSA or CT). Protein association onto the bilayer-covered polystyrene sulfate (PSS) was determined from adsorption isotherms, dynamic light scattering for size distributions and zeta-potential analysis revealing a monomolecular, thin and highly organized protein layer surrounding each particle with potential for biospecific recognition such as antigen-antibody, receptor-ligand, hybridization of oligonucleotide sequences, all of them important in immunodiagnosis, selective biomolecular chromatographic separations, microarrays design and others.

Screening the Schistosoma mansoni transcriptome for genes differentially expressed in the schistosomulum stage in search for vaccine candidates

Schistosomiasis affects more than 200 million people worldwide; another 600 million are at risk of infection. The schistosomulum stage is believed to be the target of protective immunity in the attenuated cercaria vaccine model. In an attempt to identify genes up-regulated in the schistosomulum stage in relation to cercaria, we explored the Schistosoma mansoni transcriptome by looking at the relative frequency of reads in EST libraries from both stages. The 400 genes potentially up-regulated in schistosomula were analyzed as to their Gene Ontology categorization, and we have focused on those encoding-predicted proteins with no similarity to proteins of other organisms, assuming they could be parasite-specific proteins important for survival in the host. Up-regulation in schistosomulum relative to cercaria was validated with real-time reverse transcription polymerase chain reaction (RT-PCR) for five out of nine selected genes (56%). We tested their protective potential in mice through immunization with DNA vaccines followed by a parasite challenge. Worm burden reductions of 16-17% were observed for one of them, indicating its protective potential. Our results demonstrate the value and caveats of using stage-associated frequency of ESTs as an indication of differential expression coupled to DNA vaccine screening in the identification of novel proteins to be further investigated as potential vaccine candidates.

Base Excision Repair Activities Differ in Human Lung Cancer Cells and Corresponding Normal Controls

Oxidative damage to DNA is thought to play a role in carcinogenesis by causing Mutations, and indeed accumulation of oxidized DNA bases has been observed in samples obtained from tumors but not from surrounding tissue within the same patient. Base excision repair (BER) is the main pathway for the repair of oxidized modifications both in nuclear and mitochondrial, DNA. In order to ascertain whether diminished BER capacity might account for increased levels of oxidative DNA damage in cancer cells, the activities of BER enzymes in three different lung cancer cell lines and their non-cancerous counterparts were measured using oligonucleotide substrates with single DNA lesions to assess specific BER enzymes. The activities of four BER enzymes, OGG1, NTH1, UDG and APE1, were compared in mitochondrial and nuclear extracts. For each specific lesion, the repair activities were similar among the three cell lines used. However, the specific activities and cancer versus control comparison differed significantly between the nuclear and mitochondrial compartments. OGG1 activity, as measured by 8-oxodA incision, was upregulated in cancer cell mitochondria but down-regulated in the nucleus when compared to control cells. Similarly, NTH1 activity was also up-regulated in mitochondrial extracts from cancer cells but did not change significantly in the nucleus. Together, these results support the idea that alterations in BER capacity are associated with carcinogenesis.