Antisense-mediated splice intervention to treat human disease: the odyssey continues

Recent approvals of oligonucleotide analogue drugs to alter gene expression have been welcomed by patient communities but not universally supported. These compounds represent a class of drugs that are designed to target a specific gene transcript, and they include a number of chemical entities to evoke different antisense mechanisms, depending upon the disease aetiology. To date, oligonucleotide therapeutics that are in the clinic or at advanced stages of translation target rare diseases, posing challenges to clinical trial design, recruitment and evaluation and requiring new evaluation paradigms. This review discusses the currently available and emerging therapeutics that alter exon selection through an effect on pre-mRNA splicing and explores emerging concerns over safety and efficacy. Although modification of synthetic nucleic acids destined for therapeutic application is common practice to protect against nuclease degradation and to influence drug function, such modifications may also confer unexpected physicochemical and biological properties. Negatively charged oligonucleotides have a strong propensity to bind extra- and intra-cellular proteins, whereas those analogues with a neutral backbone show inefficient cellular uptake but excellent safety profiles. In addition, the potential for incorporation of chemically modified nucleic acid monomers, yielded by nuclease degradation of exogenous oligonucleotides, into biomolecules has been raised and the possibility not entirely discounted. We conclude with a commentary on the ongoing efforts to develop novel antisense compounds and enhance oligonucleotide delivery in order to further improve efficacy and accelerate implementation of antisense therapeutics for human disease

ESR and EISCAT observations of the response of the cusp and cleft to IMF orientation changes

International audienceWe report observations of the cusp/cleft ionosphere made on December 16th 1998 by the EISCAT (European incoherent scatter) VHF radar at Tromsø and the EISCAT Svalbard radar (ESR). We compare them with observations of the dayside auroral luminosity, as seen by meridian scanning photometers at Ny Ålesund and of HF radar backscatter, as observed by the CUTLASS radar. We study the response to an interval of about one hour when the interplanetary magnetic field (IMF), monitored by the WIND and ACE spacecraft, was southward. The cusp/cleft aurora is shown to correspond to a spatially extended region of elevated electron temperatures in the VHF radar data. Initial conditions were characterised by a northward-directed IMF and cusp/cleft aurora poleward of the ESR. A strong southward turning then occurred, causing an equatorward motion of the cusp/cleft aurora. Within the equatorward expanding, southward-IMF cusp/cleft, the ESR observed structured and elevated plasma densities and ion and electron temperatures. Cleft ion fountain upflows were seen in association with elevated ion temperatures and rapid eastward convection, consistent with the magnetic curvature force on newly opened field lines for the observed negative IMF By. Subsequently, the ESR beam remained immediately poleward of the main cusp/cleft and a sequence of poleward-moving auroral transients passed over it. After the last of these, the ESR was in the polar cap and the radar observations were characterised by extremely low ionospheric densities and downward field-aligned flows. The IMF then turned northward again and the auroral oval contracted such that the ESR moved back into the cusp/cleft region. For the poleward-retreating, northward-IMF cusp/cleft, the convection flows were slower, upflows were weaker and the electron density and temperature enhancements were less structured. Following the northward turning, the bands of high electron temperature and cusp/cleft aurora bifurcated, consistent with both subsolar and lobe reconnection taking place simultaneously. The present paper describes the large-scale behaviour of the ionosphere during this interval, as observed by a powerful combination of instruments. Two companion papers, by Lockwood et al. (2000) and Thorolfsson et al. (2000), both in this issue, describe the detailed behaviour of the poleward-moving transients observed during the interval of southward Bz, and explain their morphology in the context of previous theoretical work

The effect of electromagnetic radiation in the mobile phone range on the behaviour of the rat

Abstract Electromagnetic radiation (EMR) is emitted from electromagnetic fields that surround power lines, household appliances and mobile phones. Research has shown that there are connections between EMR exposure and cancer and also that exposure to EMR may result in structural damage to neurons. In a study by Salford et al. (Environ Health Perspect 111:881-883, 2003) the authors demonstrated the presence of strongly stained areas in the brains of rats that were exposed to mobile phone EMR. These darker neurons were particularly prevalent in the hippocampal area of the brain. The aim of our study was to further investigate the effects of EMR. Since the hippocampus is involved in learning and memory and emotional states, we hypothesised that EMR will have a negative impact on the subject's mood and ability to learn. We subsequently performed behavioural, histological and biochemical tests on exposed and unexposed male and female rats to determine the effects of EMR on learning and memory, emotional states and corticosterone levels. We found no significant differences in the spatial memory test, and morphological assessment of the brain also yielded non-significant differences between the groups. However, in some exposed animals there were decreased locomotor activity, increased grooming and a tendency of increased basal corticosterone levels. These findings suggested that EMR exposure may lead to abnormal brain functioning

Characterising splicing defects of ABCA4 variants within exons 13–50 in patient-derived fibroblasts

The ATP-binding cassette subfamily A member 4 gene (ABCA4)-associated retinopathy, Stargardt disease, is the most common monogenic inherited retinal disease. Given the pathogenicity of numerous ABCA4 variants is yet to be examined and a significant proportion (more than 15%) of ABCA4 variants are categorized as splice variants in silico, we therefore established a fibroblast-based splice assay to analyze ABCA4 variants in an Australian Stargardt disease cohort and characterize the pathogenic mechanisms of ABCA4 variants. A cohort of 67 patients clinically diagnosed with Stargardt disease was recruited. Genomic DNA was analysed using a commercial panel for ABCA4 variant detection and the consequences of ABCA4 variants were predicted in silico. Dermal fibroblasts were propagated from skin biopsies, total RNA was extracted and the ABCA4 transcript was amplified by RT-PCR. Our analysis identified a total of 67 unique alleles carrying 74 unique variants. The most prevalent splice-affecting complex allele c.[5461-10T > C; 5603A > T] was carried by 10% of patients in a compound heterozygous state. ABCA4 transcripts from exon 13 to exon 50 were readily detected in fibroblasts. In this region, aberrant splicing was evident in 10 out of 57 variant transcripts (18%), carried by 19 patients (28%). Patient-derived fibroblasts provide a feasible platform for identification of ABCA4 splice variants located within exons 13–50. Experimental evidence of aberrant splicing contributes to the pathogenic classification for ABCA4 variants. Moreover, identification of variants that affect splicing processes provides opportunities for intervention, in particular antisense oligonucleotide-mediated splice correction

Virulence Potential and Genomic Mapping of the Worldwide Clone Escherichia coli ST131

Recently, the worldwide propagation of clonal CTX-M-15-producing Escherichia coli isolates, namely ST131 and O25b:H4, has been reported. Like the majority of extra-intestinal pathogenic E. coli isolates, the pandemic clone ST131 belongs to phylogenetic group B2, and has recently been shown to be highly virulent in a mouse model, even though it lacks several genes encoding key virulence factors (Pap, Cnf1 and HlyA). Using two animal models, Caenorhabditis elegans and zebrafish embryos, we assessed the virulence of three E. coli ST131 strains (2 CTX-M-15- producing urine and 1 non-ESBL-producing faecal isolate), comparing them with five non-ST131 B2 and a group A uropathogenic E. coli (UPEC). In C. elegans, the three ST131 strains showed intermediate virulence between the non virulent group A isolate and the virulent non-ST131 B2 strains. In zebrafish, the CTX-M-15-producing ST131 UPEC isolates were also less virulent than the non-ST131 B2 strains, suggesting that the production of CTX-M-15 is not correlated with enhanced virulence. Amongst the non-ST131 B2 group isolates, variation in pathogenic potential in zebrafish embryos was observed ranging from intermediate to highly virulent. Interestingly, the ST131 strains were equally persistent in surviving embryos as the non-ST131-group B2 strains, suggesting similar mechanisms may account for development of persistent infection. Optical maps of the genome of the ST131 strains were compared with those of 24 reference E. coli strains. Although small differences were seen within the ST131 strains, the tree built on the optical maps showed that these strains belonged to a specific cluster (86% similarity) with only 45% similarity with the other group B2 strains and 25% with strains of group A and D. Thus, the ST131 clone has a genetic composition that differs from other group B2 strains, and appears to be less virulent than previously suspected

Propolis can potentialise the anti-adhesion activity of proanthocyanidins on uropathogenic Escherichia coli in the prevention of recurrent urinary tract infections

<p>Abstract</p> <p>Background</p> <p><it>Escherichia coli</it>, the main bacteria found in recurrent urinary tract infections (UTI), is now frequently resistant to several currently used antibiotic treatments making new solutions essential. In this study, we evaluated the association propolis and proanthocyanidins type A to reduce bacterial anti-adhesion activity of <it>E. coli </it>on urothelial cells.</p> <p>Results</p> <p>This first double-blind, randomized, cross-over human trial included 5 volunteers that followed 6 different regimens with or without variable doses of cranberry and propolis with a washout period of at least 1 week between each regimen. Urine samples were collected at 0 h, 4-6 h, 12 h and 24 h after cranberry plus propolis or placebo capsule consumption. In vivo urinary bacterial anti-adhesion activity was assessed with a bioassay (a human T24 epithelial cell-line assay) and an in vivo <it>Caenorhabditis elegans </it>model. HPLC-PDA-MS was used to detect propolis and cranberry compounds in urine. Bioassays indicated significant bacterial anti-adhesion activity in urine collected from volunteers who had consumed cranberry plus propolis powder compared to placebo (<it>p </it>< 0.001). This inhibition was clearly dose-dependent, increasing with the amount of PACs and propolis equivalents consumed in each regimen. Results suggested that propolis had an additional effect with PACs and prevent a bacterial anti-adhesion effect over 1 day. An in vivo model showed that the <it>E. coli </it>strain presented a reduced ability to kill <it>C. elegans </it>after their growth in urine samples of patients who took cranberry plus propolis capsules. HPLC confirmed that propolis is excreted in urine.</p> <p>Conclusions</p> <p>This study presents an alternative to prevent recurrent UTI. Administration of PACs plus propolis once daily offers some protection against bacterial adhesion, bacterial multiplication and virulence in the urinary tract, representing an interesting new strategy to prevent recurrent UTI.</p

Biodegradation of ochratoxin A by Pediococcus parvulus isolated from Douro wines

Lactic acid bacteria (LAB) are a promising solution to reduce exposure to dietary mycotoxins because of the unique mycotoxin decontaminating characteristic of some LAB. Ochratoxin A (OTA) is one of the most prominent mycotoxins found in agricultural commodities. The present work reports on the ability of Pediococcus parvulus strains that were isolated from Douro wines that spontaneously underwent malolactic fermentation to detoxify OTA. These strains were identified and characterised using a polyphasic approach that employed both phenotypic and genotypic methods. When cultivated on OTA-supplemented MRS media, OTA was biodegraded into OTα by certain P. parvulus strains. The presence of OTα was confirmed using LC-MS/MS. The conversion of OTA into OTα indicates that the OTA amide bond was hydrolysed by a putative peptidase. The rate of OTA biodegradation was found to be dependent on the inoculum size and on the incubation temperature. Adsorption assays with dead P. parvulus cells showed that approximately 1.3% ± 1.0 of the OTA was adsorbed onto cells wall, which excludes this mechanism in the elimination of OTA by strains that degrades OTA. Under optimum conditions, 50% and 90% of OTA was degraded in 6 and 19 h, respectively. Other LAB strains that belonged to different species were tested but did not degrade OTA. OTA biodegradation by P. parvulus UTAD 473 was observed in grape must. Because some P. parvulus strains have relevant probiotic properties, the strains that were identified could be particularly relevant to food and feed applications to counteract the toxic effects of OTA.This work was funded by FEDER funds through the "Programa Operacional Factores de Competitividade - COMPETE" and by national funds through "Fundacao para a Ciencia e a Tecnologia - FCT", Ref. FCOMP-01-0124-FEDER-028029 and PTDC/AGR-TEC/3900/2012, respectively. The authors also thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the Project "BioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes, REF. NORTE-07-0124-FEDER-000028" co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER. Luis Abrunhosa was supported by the grant SFRH/BPD/43922/2008 from FCT