Redesigned and chemically-modified hammerhead ribozymes with improved activity and serum stability

BACKGROUND: Hammerhead ribozymes are RNA-based molecules which bind and cleave other RNAs specifically. As such they have potential as laboratory reagents, diagnostics and therapeutics. Despite having been extensively studied for 15 years or so, their wide application is hampered by their instability in biological media, and by the poor translation of cleavage studies on short substrates to long RNA molecules. This work describes a systematic study aimed at addressing these two issues. RESULTS: A series of hammerhead ribozyme derivatives, varying in their hybridising arm length and size of helix II, were tested in vitro for cleavage of RNA derived from the carbamoyl phosphate synthetase II gene of Plasmodium falciparum. Against a 550-nt transcript the most efficient (t(1/2 )= 26 seconds) was a miniribozyme with helix II reduced to a single G-C base pair and with twelve nucleotides in each hybridising arm. Miniribozymes of this general design were targeted to three further sites, and they demonstrated exceptional cleavage activity. A series of chemically modified derivatives was prepared and examined for cleavage activity and stability in human serum. One derivative showed a 10(3)-fold increase in serum stability and a doubling in cleavage efficiency compared to the unmodified miniribozyme. A second was almost 10(4)-fold more stable and only 7-fold less active than the unmodified parent. CONCLUSION: Hammerhead ribozyme derivatives in which helix II is reduced to a single G-C base pair cleave long RNA substrates very efficiently in vitro. Using commonly available phosphoramidites and reagents, two patterns of nucleotide substitution in this derivative were identified which conferred both good cleavage activity against long RNA targets and good stability in human serum

Entomological indices of malaria transmission in Chikhwawa district, Southern Malawi

Abstract Background Although malaria is highly prevalent throughout Malawi, little is known of its transmission dynamics. This paper describes the seasonal activity of the different vectors, human biting indices, sporozoite rates and the entomological inoculation rate in a low-lying rural area in southern Malawi. Methods Vectors were sampled over 52 weeks from January 2002 to January 2003, by pyrethrum knockdown catch in two villages in Chikhwawa district, in the Lower Shire Valley. Results In total, 7,717 anophelines were collected of which 55.1% were Anopheles gambiae sensu lato and 44.9% were Anopheles funestus. Three members of the An. gambiae complex were identified by PCR: Anopheles arabiensis (75%) was abundant throughout the year, An. gambiae s.s. (25%) was most common during the wet season and Anopheles quadriannulatus occurred at a very low frequency (n=16). An. funestus was found in all samples but was most common during the dry season. Anopheles gambiae s.s. and An. funestus were highly anthropophilic with human blood indices of 99.2% and 96.3%, respectively. Anopheles arabiensis had fed predominantly on humans (85.0%) and less commonly on cattle (10.9%; 1.2% of blood meals were of mixed origin). Plasmodium falciparum (192/3,984) and Plasmodium malariae (1/3,984) sporozoites were detected by PCR in An. arabiensis (3.2%) and An. funestus (4.5%), and in a significantly higher proportion of An. gambiae s.s. (10.6%)(pP. falciparum sporozoite rate was 4.8%, resulting in estimated inoculation rates of 183 infective bites/ person per annum, or an average rate of ~15 infective bites/person/month. Conclusions The results demonstrate the importance of An. gambiae s.s., An. arabiensis and An. funestus in driving the high levels of malaria transmission in the south of Malawi. Sustained and high coverage or roll out of current approaches to malaria control (primarily insecticide-treated bed nets and indoor residual house spraying) in the area are likely to reduce the observed high malaria transmission rate and consequently the incidence of human infections, unless impeded by increasing resistance of vectors to insecticides.</p

Diffuse retro-reflective imaging for improved mosquito tracking around human baited bednets

Robust imaging techniques for tracking insects have been essential tools in numerous laboratory and field studies on pests, beneficial insects and model systems. Recent innovations in optical imaging systems and associated signal processing have enabled detailed characterisation of nocturnal mosquito behaviour around bednets and improvements in bednet design, a global essential for protecting populations against malaria. Nonetheless, there remain challenges around ease of use for large scale in situ recordings and extracting data reliably in the critical areas of the bednet where the optical signal is attenuated. Here we introduce a retro-reflective screen at the back of the measurement volume, which can simultaneously provide diffuse illumination, and remove optical alignment issues whilst requiring only one-sided access to the measurement space. The illumination becomes significantly more uniform, although, noise removal algorithms are needed to reduce the effects of shot noise particularly across low intensity bednet regions. By systematically introducing mosquitoes in front and behind the bednet in lab experiments we are able to demonstrate robust tracking in these challenging areas. Overall, the retro-reflective imaging setup delivers mosquito segmentation rates in excess of 90% compared to less than 70% with back-lit systems

B-type natriuretic peptide predicts deterioration in functional capacity following lung resection

OBJECTIVES Following lung resection, there is a decrease in the functional capacity and quality of life, which is not fully explained by changes in pulmonary function. Previous work demonstrates that B-type natriuretic peptide (BNP) is associated with short- and long-term complications following lung resection, leading to the suggestion that cardiac dysfunction may contribute to functional deterioration. Our aim was to investigate any relationship between BNP and subjective and objective indices of functional deterioration following lung resection surgery. METHODS Twenty-seven patients undergoing lung resection had serum BNP measured preoperatively, on postoperative day (POD)1 and POD2, and at 2 months postoperatively. The functional deterioration was assessed using 6-min walk tests and the Medical Research Council dyspnoea scale. ‘Deterioration in functional capacity’ was defined as either an increase in the Medical Research Council dyspnoea score or a significant decrease in the 6-min walk test distance. RESULTS BNP increased over time (P &lt; 0.01) and was significantly elevated on POD1 and POD2 (P &lt; 0.02 for both). Seventeen patients demonstrated functional deterioration 2 months postoperatively. At all perioperative time points, BNP was significantly higher in patients showing deterioration (P &lt; 0.05 for all). Preoperative BNP was predictive of functional deterioration at 2 months with an area under the receiver-operating characteristic curve of 0.82 (P = 0.01, 95% confidence interval 0.65–0.99). CONCLUSIONS This study has demonstrated, using subjective and objective measures, that preoperative BNP is a predictor of functional deterioration following lung resection. BNP may have a role in preoperative risk stratification in this population, allowing therapy in future to be targeted towards high-risk patients with the aim of preventing postoperative cardiac dysfunction. Clinical trial registration number: NCT01892800

Effective autodissemination of pyriproxyfen to breeding sites by the exophilic malaria vector Anopheles arabiensis in semi-field settings in Tanzania

BACKGROUND Malaria vector control strategies that target adult female mosquitoes are challenged by the emergence of insecticide resistance and behavioural resilience. Conventional larviciding is restricted by high operational costs and inadequate knowledge of mosquito-breeding habitats in rural settings that might be overcome by the juvenile hormone analogue, Pyriproxyfen (PPF). This study assessed the potential for Anopheles arabiensis to pick up and transfer lethal doses of PPF from contamination sites to their breeding habitats (i.e. autodissemination of PPF). METHODS A semi-field system (SFS) with four identical separate chambers was used to evaluate PPF-treated clay pots for delivering PPF to resting adult female mosquitoes for subsequent autodissemination to artificial breeding habitats within the chambers. In each chamber, a tethered cow provided blood meals to laboratory-reared, unfed female An. arabiensis released in the SFS. In PPF-treated chambers, clay pot linings were dusted with 0.2 - 0.3 g AI PPF per pot. Pupae were removed from the artificial habitats daily, and emergence rates calculated. Impact of PPF on emergence was determined by comparing treatment with an appropriate control group. RESULTS Mean (95%CI) adult emergence rates were (0.21 +/- 0.299) and (0.95 +/- 0.39) from PPF-treated and controls respectively (p < 0.0001). Laboratory bioassay of water samples from artificial habitats in these experiments resulted in significantly lower emergence rates in treated chambers (0.16 +/- 0.23) compared to controls 0.97 +/- 0.05) (p < 0.0001). In experiments where no mosquitoes introduced, there were no significant differences between control and treatment, indicating that transfer of PPF to breeding sites only occurred when mosquitoes were present; i.e. that autodissemination had occurred. Treatment of a single clay pot reduced adult emergence in six habitats to (0.34 +/- 0.13) compared to (0.98 +/- 0.02) in the controls (p < 0.0001), showing a high level of habitats coverage amplification of the autodissemination event. CONCLUSION The study provides proof of principle for the autodissemination of PPF to breeding habitats by malaria vectors. These findings highlight the potential for this technique for outdoor control of malaria vectors and call for the testing of this technique in field trials

Infrared video tracking of Anopheles gambiae at insecticide-treated bed nets reveals rapid decisive impact after brief localised net contact

Long-lasting insecticidal bed nets (LLINs) protect humans from malaria transmission and are fundamental to malaria control worldwide, but little is known of how mosquitoes interact with nets. Elucidating LLIN mode of action is essential to maintain or improve efficacy, an urgent need as emerging insecticide resistance threatens their future. Tracking multiple free-flying Anopheles gambiae responding to human-occupied bed nets in a novel large-scale system, we characterised key behaviours and events. Four behavioural modes with different levels of net contact were defined: swooping, visiting, bouncing and resting. Approximately 75% of all activity occurred at the bed net roof where multiple brief contacts were focussed above the occupant’s torso. Total flight and net contact times were lower at LLINs than untreated nets but the essential character of the response was unaltered. LLINs did not repel mosquitoes but impacted rapidly: LLIN contact of less than 1 minute per mosquito during the first ten minutes reduced subsequent activity; after thirty minutes, activity at LLINs was negligible. Velocity measurements showed that mosquitoes detected nets, including unbaited untreated nets, prior to contact. This is the most complete characterisation of mosquito-LLIN interactions to date, and reveals many aspects of LLIN mode of action, important for developing the next generation of LLINs

Evaluation of the vector competence of a native UK mosquito Ochlerotatus detritus (Aedes detritus) for dengue, chikungunya and West Nile viruses

BACKGROUND: To date there has been no evidence of mosquito-borne virus transmission of public health concern in the UK, despite the occurrence of more than 30 species of mosquito, including putative vectors of arboviruses. The saltmarsh mosquito Ochlerotatus detritus [syn. Aedes (Ochlerotatus) detritus] is locally common in parts of the UK where it can be a voracious feeder on people. METHODS: Here, we assess the competence of O. detritus for three major arboviruses: dengue virus (DENV), chikungunya virus (CHIKV) and West Nile virus (WNV) using adult mosquitoes reared from wild, field-obtained immatures. RESULTS: We demonstrate laboratory competence for WNV at 21 °C, with viral RNA detected in the mosquito’s saliva 17 days after oral inoculation. By contrast, there was no evidence of laboratory competence of O. detritus for either DENV or CHIKV. CONCLUSIONS: To our knowledge, this is the first study to demonstrate competence of a UK mosquito for WNV and confirms that O. detritus may present a potential risk for arbovirus transmission in the UK and that further investigation of its vector role in the wild is required