Improving gene silencing of siRNAs via tricyclo-DNA modification

;Small interfering RNAs (siRNAs) can be exploited for the selective silencing of disease-related genes via the RNA interference (RNAi) machinery and therefore raise hope for future therapeutic applications. Especially chemically modified siRNAs are of interest as they are expected to convert lead siRNA sequences into effective drugs. To study the potential of tricyclo-DNA (tc-DNA) in this context we systematically incorporated tc-DNA units at various positions in a siRNA duplex targeted to the EGFP gene that was expressed in HeLa cells. Silencing activity was measured by FACS, mRNA levels were determined by RT-PCR and the biostability of the modifed siRNAs was determined in human serum. We found that modifications in the 3'-overhangs in both the sense and antisense strands were compatible with the RNAi machinery leading to similar activities compared to wild type (wt) siRNA. Additional modifications at the 3'-end, the 5'- end and in the center of the sense (passenger) strand were also well tolerated and did not compromise activity. Extensive modifications of the 3'- and the 5'-end in the antisense (guide) strand, however, abolished RNAi activity. Interestingly, modifications in the center of the duplex on both strands, corresponding to the position of the cleavage site by AGO2, increased efficacy relative to wt by a factor of 4 at the lowest concentrations (2 nM) investigated. In all cases, reduction of EGFP fluorescence was accompanied with a reduction of the EGFP mRNA level. Serum stability analysis further showed that 3'-overhang modifications only moderately increased stability while more extensive substitution by tc-DNA residues significantly enhanced biostability.

Screening the Structural Space of Bicyclo-DNA: Synthesis and Properties of Bicyclo-DNA Functionalized at C(6’)

The synthesis of a novel bicyclo-thymidine nucleoside bearing an ester functionality at C(6') (bc(alpha-alk)-nucleosides) is reported. This nucleoside was incorporated into oligodeoxynucleotides via solid phase phosphoramidite chemistry, and the ester moiety was post-synthetically converted to an amide or a carboxy group, or was left unchanged. Thermal melting data (T-m) with complementary DNA and RNA were collected and compared to natural DNA and to bc- and bc(ox)-DNA. It was found that single incorporations of bc(alpha-alk)-nucleosides in DNA duplexes were destabilizing by 0.5 to 2.5 degrees C/mod, whereas two consecutive bc(alpha-alk)-residues were less destabilizing, and in some cases even stabilizing by 0.5 degrees C/mod. In duplexes with complementary RNA, isolated bc(alpha-alk)-residues destabilized the duplex by -1.0 to -4.0 degrees C/mod, depending on the chemical nature of the substituent, whereas two consecutive modifications were only destabilizing by 0.3-1.0 degrees C/mod. The pairing selectivity was similar to that of unmodified or bc-DNA.

Hydrogen bonding of nucleotide base pairs: Application of the PM3 method

The ability of the pm3 semiempirical quantum mechanical method to reproduce hydrogen bonding in nucleotide base pairs was assessed. Results of pm3 calculations on the nucleotides 2′-deoxyadenosine 5′-monophosphate (pdA), 2′-deoxyguanosine 5′-monophosphate (pdG), 2′-deoxycytidine 5′-monophosphate (pdC), and 2′-deoxythymidine 5′-monophosphate (pdT) and the base pairs pdA–pdT, pdG–pdC, and pdG(syn)–pdC are presented and discussed. The pm3 method is the first of the parameterized nddo quantum mechanical models with any ability to reproduce hydrogen bonding between nucleotide base pairs. Intermolecular hydrogen bond lengths between nucleotides displaying Watson–Crick base pairing are 0.1–0.2 Å less than experimental results. Nucleotide bond distances, bond angles, and torsion angles about the glycosyl bond (χ), the C4′C5′ bond (γ), and the C5′O5′ bond (β) agree with experimental results. There are many possible conformations of nucleotides. pm3 calculations reveal that many of the most stable conformations are stabilized by intramolecular CHO hydrogen bonds. These interactions disrupt the usual sugar puckering. The stacking interactions of a dT–pdA duplex are examined at different levels of gradient optimization. The intramolecular hydrogen bonds found in the nucleotide base pairs disappear in the duplex, as a result of the additional constraints on the phosphate group when part of a DNA backbone. Sugar puckering is reproduced by the pm3 method for the four bases in the dT–pdA duplex. pm3 underestimates the attractive stacking interactions of base pairs in a B-DNA helical conformation. The performance of the pm3 method implemented in SPARTAN is contrasted with that implemented in MOPAC. At present, accurate ab initio calculations are too timeconsuming to be of practical use, and molecular mechanics methods cannot be used to determine quantum mechanical properties such as reaction-path calculations, transition-state structures, and activation energies. The pm3 method should be used with extreme caution for examination of small DNA systems. Future parameterizations of semiempirical methods should incorporate base stacking interactions into the parameterization data set to enhance the ability of these methods.

Chapter VI: Follow-up after revascularisation

Structured follow-up after revascularisation for chronic critical limb ischaemia (CLI) aims at sustained treatment success and continued best patient care. Thereby, efforts need to address three fundamental domains: (A) best medical therapy, both to protect the arterial reconstruction locally and to reduce atherosclerotic burden systemically; (B) surveillance of the arterial reconstruction; and (C) timely initiation of repeat interventions. As most CLI patients are elderly and frail, sustained resolution of CLI and preserved ambulatory capacity may decide over independent living and overall prognosis. Despite this importance, previous guidelines have largely ignored follow-up after CLI; arguably because of a striking lack of evidence and because of a widespread assumption that, in the context of CLI, efficacy of initial revascularisation will determine prognosis during the short remaining life expectancy. This chapter of the current CLI guidelines aims to challenge this disposition and to recommend evidentially best clinical practice by critically appraising available evidence in all of the above domains, including antiplatelet and antithrombotic therapy, clinical surveillance, use of duplex ultrasound, and indications for and preferred type of repeat interventions for failing and failed reconstructions. However, as corresponding studies are rarely performed among CLI patients specifically, evidence has to be consulted that derives from expanded patient populations. Therefore, most recommendations are based on extrapolations or subgroup analyses, which leads to an almost systematic degradation of their strength. Endovascular reconstruction and surgical bypass are considered separately, as are specific contexts such as diabetes or renal failure; and critical issues are highlighted throughout to inform future studies.

Chapter II: Diagnostic methods

Non-invasive vascular studies can provide crucial information on the presence, location, and severity of critical limb ischaemia (CLI), as well as the initial assessment or treatment planning. Ankle-brachial index with Doppler ultrasound, despite limitations in diabetic and end-stage renal failure patients, is the first-line evaluation of CLI. In this group of patients, toe-brachial index measurement may better establish the diagnosis. Other non-invasive measurements, such as segmental limb pressure, continuous-wave Doppler analysis and pulse volume recording, are of limited accuracy. Transcutaneous oxygen pressure (TcPO(2)) measurement may be of value when rest pain and ulcerations of the foot are present. Duplex ultrasound is the most important non-invasive tool in CLI patients combining haemodynamic evaluation with imaging modality. Computed tomography angiography (CTA) and magnetic resonance angiography (MRA) are the next imaging studies in the algorithm for CLI. Both CTA and MRA have been proven effective in aiding the decision-making of clinicians and accurate planning of intervention. The data acquired with CTA and MRA can be manipulated in a multiplanar and 3D fashion and can offer exquisite detail. CTA results are generally equivalent to MRA, and both compare favourably with contrast angiography. The individual use of different imaging modalities depends on local availability, experience, and costs. Contrast angiography represents the gold standard, provides detailed information about arterial anatomy, and is recommended when revascularisation is needed.

Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

Consensus Document of the International Union of Angiology (IUA)-2013. Current concept on the management of arterio-venous management

Arterio-venous malformations (AVMs) are congenital vascular malformations (CVMs) that result from birth defects involving the vessels of both arterial and venous origins, resulting in direct communications between the different size vessels or a meshwork of primitive reticular networks of dysplastic minute vessels which have failed to mature to become 'capillary' vessels termed "nidus". These lesions are defined by shunting of high velocity, low resistance flow from the arterial vasculature into the venous system in a variety of fistulous conditions. A systematic classification system developed by various groups of experts (Hamburg classification, ISSVA classification, Schobinger classification, angiographic classification of AVMs,) has resulted in a better understanding of the biology and natural history of these lesions and improved management of CVMs and AVMs. The Hamburg classification, based on the embryological differentiation between extratruncular and truncular type of lesions, allows the determination of the potential of progression and recurrence of these lesions. The majority of all AVMs are extra-truncular lesions with persistent proliferative potential, whereas truncular AVM lesions are exceedingly rare. Regardless of the type, AV shunting may ultimately result in significant anatomical, pathophysiological and hemodynamic consequences. Therefore, despite their relative rarity (10-20% of all CVMs), AVMs remain the most challenging and potentially limb or life-threatening form of vascular anomalies. The initial diagnosis and assessment may be facilitated by non- to minimally invasive investigations such as duplex ultrasound, magnetic resonance imaging (MRI), MR angiography (MRA), computerized tomography (CT) and CT angiography (CTA). Arteriography remains the diagnostic gold standard, and is required for planning subsequent treatment. A multidisciplinary team approach should be utilized to integrate surgical and non-surgical interventions for optimum care. Currently available treatments are associated with significant risk of complications and morbidity. However, an early aggressive approach to elimiate the nidus (if present) may be undertaken if the benefits exceed the risks. Trans-arterial coil embolization or ligation of feeding arteries where the nidus is left intact, are incorrect approaches and may result in proliferation of the lesion. Furthermore, such procedures would prevent future endovascular access to the lesions via the arterial route. Surgically inaccessible, infiltrating, extra-truncular AVMs can be treated with endovascular therapy as an independent modality. Among various embolo-sclerotherapy agents, ethanol sclerotherapy produces the best long term outcomes with minimum recurrence. However, this procedure requires extensive training and sufficient experience to minimize complications and associated morbidity. For the surgically accessible lesions, surgical resection may be the treatment of choice with a chance of optimal control. Preoperative sclerotherapy or embolization may supplement the subsequent surgical excision by reducing the morbidity (e.g. operative bleeding) and defining the lesion borders. Such a combined approach may provide an excellent potential for a curative result. Conclusion. AVMs are high flow congenital vascular malformations that may occur in any part of the body. The clinical presentation depends on the extent and size of the lesion and can range from an asymptomatic birthmark to congestive heart failure. Detailed investigations including duplex ultrasound, MRI/MRA and CT/CTA are required to develop an appropriate treatment plan. Appropriate management is best achieved via a multi-disciplinary approach and interventions should be undertaken by appropriately trained physicians.

Surveillance after distal revascularization for critical limb ischaemia

Patients with critical limb ischaemia (CLI) are usually elderly and suffer from several co-morbidities. The goal of surveillance after both endovascular and surgical revascularization for CLI is not only the protection of re-established distal perfusion and sustained ambulation but also the reduction of systemic atherothrombotic risk and mortality by ensuring continued best medical care. However, preferred format and rhythm of structured follow-up programs have remained controversial, mainly because of lack of compelling evidence. This review aims to summarize and to appraise available information critically. Thereby, it underlines the importance of systematic surveillance after both surgical and endovascular revascularization for CLI. Recent European guidelines are considered and areas of uncertainty are highlighted and discussed. According to currently available literature and recent guidelines, the early duplex scan is justified in all patients undergoing endovascular or surgical distal revascularization for CLI. There is no best level evidence supporting continued long term duplex surveillance of revascularizations with normal findings at early duplex scan, whereas those patients with abnormal early duplex scan or high risk revacularization are likely to benefit from continued duplex surveillance. Regular clinical follow-up is suggested and clinical deterioration should trigger duplex scanning to ensure revascularization patency.

A modified calculation of ankle-brachial pressure index is far more sensitive in the detection of peripheral arterial disease

BACKGROUND: Ankle-brachial pressure index (ABI) is a simple, inexpensive, and useful tool in the detection of peripheral arterial occlusive disease (PAD). The current guidelines published by the American Heart Association define ABI as the quotient of the higher of the systolic blood pressures (SBPs) of the two ankle arteries of that limb (either the anterior tibial artery or the posterior tibial artery) and the higher of the two brachial SBPs of the upper limbs. We hypothesized that considering the lower of the two ankle arterial SBPs of a side as the numerator and the higher of the brachial SBPs as the denominator would increase its diagnostic yield. METHODS: The former method of eliciting ABI was termed as high ankle pressure (HAP) and the latter low ankle pressure (LAP). ABI was assessed in 216 subjects and calculated according to the HAP and the LAP method. ABI findings were confirmed by arterial duplex ultrasonography. A significant arterial stenosis was assumed if ABI was <0.9. RESULTS: LAP had a sensitivity of 0.89 and a specificity of 0.93. The HAP method had a sensitivity of 0.68 and a specificity of 0.99. McNemar's test to compare the results of both methods demonstrated a two-tailed P < .0001, indicating a highly significant difference between both measurement methods. CONCLUSIONS: LAP is the superior method of calculating ABI to identify PAD. This result is of great interest for epidemiologic studies applying ABI measurements to detect PAD and assessing patients' cardiovascular risk.

Initial clinical experience with the 4-F self-expanding XPERT stent system for infrapopliteal treatment of patients with severe claudication and critical limb ischemia

PURPOSE: To evaluate the primary success and short-term patency associated with a new 4-F sheath-compatible self-expanding nitinol stent after failed conventional angioplasty of distal popliteal and infrapopliteal lesions in severe lifestyle-limiting claudication (LLC) and chronic critical limb ischemia (CLI). MATERIALS AND METHODS: Between May 2003 and July 2005, 35 patients with Rutherford category 3-5 disease (16 patients with CLI, 19 patients with LLC) underwent percutaneous transluminal angioplasty (PTA) and stent implantation. Indications for stent placement were residual stenosis, flow-limiting dissections, or elastic recoil after PTA. Before and after the intervention and during the 6-month follow-up, clinical investigation, color-flow and duplex Doppler ultrasonography, and digital subtraction angiography were performed. Technical success, primary patency at 6 months, clinical improvement as defined by Rutherford with clinical and hemodynamic measures, and complications were evaluated. RESULTS: A total of 22 patients underwent distal popliteal artery stent placement and 13 underwent tibioperoneal artery stent placement. Stent implantation was successfully performed in all patients. After stent placement, the primary cumulative patency rate for the study group at 6 months was 82%. The mean resting ankle-brachial index at baseline was 0.50 +/- 0.16 and significantly increased to 0.90 +/- 0.17 at 12-24 hours after intervention and 0.82 +/- 0.24 at latest follow-up (P < .001 for both). The sustained clinical improvement rate was 80% at the 6-month follow-up. The 6-month limb salvage rate regarding major amputation was 100%. The rate of major complications was 17%. CONCLUSIONS: Infrapopliteal application of the new nitinol stent is a safe, feasible, and effective method with good short-term patency rate in the treatment of severe LLC and chronic CLI.

Ultrasound diagnosis of spontaneous carotid dissection with isolated Horner syndrome

BACKGROUND AND PURPOSE: Isolated Horner syndrome without associated cranial nerve palsies or ischemic symptoms is an important presentation of spontaneous internal carotid artery dissection (sICAD). Ultrasound is often used as a screening method in these patients because cervical MRI is not always available on an emergency basis. Current knowledge on ultrasound findings in patients with sICAD presenting with isolated Horner syndrome is limited. METHODS: Patients were recruited from prospective cervical artery dissection databases of 3 tertiary care centers. Diagnosis of sICAD was confirmed by cervical MRI and MR angiography or digital subtraction angiography in all patients. Data on Doppler sonography and color duplex sonography examinations performed within 30 days of symptom onset were analyzed. RESULTS: We identified 88 patients with Horner syndrome as the only sign of sICAD. Initial ultrasound examination was performed in 72 patients after a mean time interval from symptom onset to examination of 11 (SD 8) days. The overall frequency of false-negative ultrasound findings was 31% (22 of 72 patients). It showed stenosis >or=80% or occlusion in 34 (47%) patients, and stenosis

Stenting of common iliac vein obstructions combined with regional thrombolysis and thrombectomy in acute deep vein thrombosis

OBJECTIVES: To evaluate the efficacy of stent placement after infrainguinal loco-regional thrombolysis and iliac thrombectomy (surgical TT) of acute deep vein thrombosis (DVT) in patients with May-Thurner-Syndrome. MATERIAL AND METHODS: We retrospectively analysed a group of 11 patients (9 women) (mean age 34 years, range 16-64 years) with surgical TT and additional intra-operative stenting due to compression of the common iliac vein. Patients underwent venography to demonstrate outflow patency after surgical TT, and to identify any obstruction at the level of the left-sided common iliac vein ("Beckenvenen-Sporn"). Obstruction at the level of arterial crossing was treated using Wallstents placed via an introducer sheath from the inguinal access site. Stents were fully deployed using balloons adjusted to the size of vein. Patients were treated with oral anticoagulants for 6 months, and followed using duplex ultrasonography. RESULTS: Technical success defined as complete vein patency and normal valve function was documented in all 11 patients. One patient needed early stent extension due to residual stenosis. At 6 months follow-up one patient (9%) had an asymptomatic occlusion of the stented common iliac vein. In all 11/11 (100%) patients the femoral segment was found to be patent, and in 1/11 (9%) there was mild reflux with few clinical symptoms of post-thrombotic syndrome. The calculated cumulative primary patency rate for venous iliac stents was 82%, and assisted patency rate was 91%, which remained unchanged over a mean follow-up of 22 months. CONCLUSION: Combining surgical TT and stenting of common iliac vein obstructions in DVT is safe, effective, and results in a acceptable venous patency.

Spectroscopic properties of pyrene-containing DNA mimics

DNA mimics containing non-nucleosidic pyrene building blocks are described. The modified oligomers form stable hybrids, although a slight reduction in hybrid stability is observed in comparison to the unmodified DNA duplex. The nature of the interaction between the pyrene residues in single and double stranded oligomers is analyzed spectroscopically. Intra- and inter-strand stacking interactions of pyrenes are monitored by UV-absorbance as well as fluorescence spectroscopy. Excimer formation is observed in both single and double strands. In general, intrastrand excimers show fluorescence emission at shorter wavelengths (approx. 5-10 nm) than excimers formed by interstrand interactions. The existence of two different forms of excimers (intra- vs. interstrand) is also revealed in temperature dependent UV-absorbance spectra. (C) 2007 Elsevier Ltd. All rights reserved.

Clinical validation of an automated vessel-segmentation software of the extracranial-carotid arteries based on 3D-MRA: A prospective study

OBJECTIVES: To determine the accuracy of automated vessel-segmentation software for vessel-diameter measurements based on three-dimensional contrast-enhanced magnetic resonance angiography (3D-MRA). METHOD: In 10 patients with high-grade carotid stenosis, automated measurements of both carotid arteries were obtained with 3D-MRA by two independent investigators and compared with manual measurements obtained by digital subtraction angiography (DSA) and 2D maximum-intensity projection (2D-MIP) based on MRA and duplex ultrasonography (US). In 42 patients undergoing carotid endarterectomy (CEA), intraoperative measurements (IOP) were compared with postoperative 3D-MRA and US. RESULTS: Mean interoperator variability was 8% for measurements by DSA and 11% by 2D-MIP, but there was no interoperator variability with the automated 3D-MRA analysis. Good correlations were found between DSA (standard of reference), manual 2D-MIP (rP=0.6) and automated 3D-MRA (rP=0.8). Excellent correlations were found between IOP, 3D-MRA (rP=0.93) and US (rP=0.83). CONCLUSION: Automated 3D-MRA-based vessel segmentation and quantification result in accurate measurements of extracerebral-vessel dimensions.

Bluetooth Enabled Ad-hoc Networks: Performance Evaluation of a Self-healing Scatternet Formation Protocol

Bluetooth wireless technology is a robust short-range communications system designed for low power (10 meter range) and low cost. It operates in the 2.4 GHz Industrial Scientific Medical (ISM) band and it employs two techniques for minimizing interference: a frequency hopping scheme which nominally splits the 2.400 - 2.485 GHz band in 79 frequency channels and a time division duplex (TDD) scheme which is used to switch to a new frequency channel on 625 μs boundaries. During normal operation a Bluetooth device will be active on a different frequency channel every 625 μs, thus minimizing the chances of continuous interference impacting the performance of the system. The smallest unit of a Bluetooth network is called a piconet, and can have a maximum of eight nodes. Bluetooth devices must assume one of two roles within a piconet, master or slave, where the master governs quality of service and the frequency hopping schedule within the piconet and the slave follows the master’s schedule. A piconet must have a single master and up to 7 active slaves. By allowing devices to have roles in multiple piconets through time multiplexing, i.e. slave/slave or master/slave, the Bluetooth technology allows for interconnecting multiple piconets into larger networks called scatternets. The Bluetooth technology is explored in the context of enabling ad-hoc networks. The Bluetooth specification provides flexibility in the scatternet formation protocol, outlining only the mechanisms necessary for future protocol implementations. A new protocol for scatternet formation and maintenance - mscat - is presented and its performance is evaluated using a Bluetooth simulator. The free variables manipulated in this study include device activity and the probabilities of devices performing discovery procedures. The relationship between the role a device has in the scatternet and it’s probability of performing discovery was examined and related to the scatternet topology formed. The results show that mscat creates dense network topologies for networks of 30, 50 and 70 nodes. The mscat protocol results in approximately a 33% increase in slaves/piconet and a reduction of approximately 12.5% of average roles/node. For 50 node scenarios the set of parameters which creates the best determined outcome is unconnected node inquiry probability (UP) = 10%, master node inquiry probability (MP) = 80% and slave inquiry probability (SP) = 40%. The mscat protocol extends the Bluetooth specification for formation and maintenance of scatternets in an ad-hoc network.