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

Heterologous prime-boost-boost immunisation of Chinese cynomolgus macaques using DNA and recombinant poxvirus vectors expressing HIV-1 virus-like particles

Background: There is renewed interest in the development of poxvirus vector-based HIV vaccines due to the protective effect observed with repeated recombinant canarypox priming with gp120 boosting in the recent Thai placebo-controlled trial. This study sought to investigate whether a heterologous prime-boost-boost vaccine regimen in Chinese cynomolgus macaques with a DNA vaccine and recombinant poxviral vectors expressing HIV virus-like particles bearing envelopes derived from the most prevalent clades circulating in sub-Saharan Africa, focused the antibody response to shared neutralising epitopes. Methods: Three Chinese cynomolgus macaques were immunised via intramuscular injections using a regimen composed of a prime with two DNA vaccines expressing clade A Env/clade B Gag followed by boosting with recombinant fowlpox virus expressing HIV-1 clade D Gag, Env and cholera toxin B subunit followed by the final boost with recombinant modified vaccinia virus Ankara expressing HIV-1 clade C Env, Gag and human complement protein C3d. We measured the macaque serum antibody responses by ELISA, enumerated T cell responses by IFN-gamma ELISpot and assessed seroneutralisation of HIV-1 using the TZM-bl beta-galactosidase assay with primary isolates of HIV-1. Results: This study shows that large and complex synthetic DNA sequences can be successfully cloned in a single step into two poxvirus vectors: MVA and FPV and the recombinant poxviruses could be grown to high titres. The vaccine candidates showed appropriate expression of recombinant proteins with the formation of authentic HIV virus-like particles seen on transmission electron microscopy. In addition the b12 epitope was shown to be held in common by the vaccine candidates using confocal immunofluorescent microscopy. The vaccine candidates were safely administered to Chinese cynomolgus macaques which elicited modest T cell responses at the end of the study but only one out of the three macaques elicited an HIV-specific antibody response. However, the antibodies did not neutralise primary isolates of HIV-1 or the V3-sensitive isolate SF162 using the TZM-bl b-galactosidase assay. Conclusions: MVA and FP9 are ideal replication-deficient viral vectors for HIV-1 vaccines due to their excellent safety profile for use in humans. This study shows this novel prime-boost-boost regimen was poorly immunogenic in Chinese cynomolgus macaques

The Female Athlete Body (FAB) Study: Rationale, Design, and Baseline Characteristics

Background: Eating Disorders (EDs) are serious psychiatric illnesses marked by psychiatric comorbidity, medical complications, and functional impairment. Research indicates that female athletes are often at greater risk for developing ED pathology versus non-athlete females. The Female Athlete Body (FAB) study is a three-site, randomized controlled trial (RCT) designed to assess the efficacy of a behavioral ED prevention program for female collegiate athletes when implemented by community providers. This paper describes the design, intervention, and participant baseline characteristics. Future papers will discuss outcomes. Methods: Female collegiate athletes (N = 481) aged 17–21 were randomized by site, team, and sport type to either FAB or a waitlist control group. FAB consisted of three sessions (1.3 h each) of a behavioral ED prevention program. Assessments were conducted at baseline (pre-intervention), post-intervention (3 weeks), and six-, 12-, and 18-month follow-ups. Results: This study achieved 96% (N = 481) of target recruitment (N = 500). Few group differences emerged at baseline. Total sample analyses revealed moderately low baseline instances of ED symptoms and clinical cases. Conclusions: Health risks associated with EDs necessitate interventions for female athletes. The FAB study is the largest existing RCT for female athletes aimed at both reduction of ED risk factors and ED prevention. The methods presented and population recruited for this study represent an ideal intervention for assessing the effects of FAB on both the aforementioned outcomes. We anticipate that findings of this study (reported in future papers) will make a significant contribution to the ED risk factor reduction and prevention literature

Sociodemographic and Built Environment Associates of Travel to School by Car among New Zealand Adolescents: Meta-Analysis.

Travelling to school by car diminishes opportunities for physical activity and contributes to traffic congestion and associated noise and air pollution. This meta-analysis examined sociodemographic characteristics and built environment associates of travelling to school by car compared to using active transport among New Zealand (NZ) adolescents. Four NZ studies (2163 adolescents) provided data on participants' mode of travel to school, individual and school sociodemographic characteristics, distance to school and home-neighbourhood built-environment features. A one-step meta-analysis using individual participant data was performed in SAS. A final multivariable model was developed using stepwise logistic regression. Overall, 60.6% of participants travelled to school by car. When compared with active transport, travelling to school by car was positively associated with distance to school. Participants residing in neighbourhoods with high intersection density and attending medium deprivation schools were less likely to travel to school by car compared with their counterparts. Distance to school, school level deprivation and low home neighbourhood intersection density are associated with higher likelihood of car travel to school compared with active transport among NZ adolescents. Comprehensive interventions focusing on both social and built environment factors are needed to reduce car travel to school

Extensive palaeo-surfaces beneath the Evans–Rutford region of the West Antarctic Ice Sheet control modern and past ice flow

The subglacial landscape of Antarctica records and influences the behaviour of its overlying ice sheet. However, in many places, the evolution of the landscape and its control on ice sheet behaviour have not been investigated in detail. Using recently released radio-echo sounding data, we investigate the subglacial landscape of the Evans–Rutford region of West Antarctica. Following quantitative analysis of the landscape morphology under ice-loaded and ice-unloaded conditions, we identify 10 flat surfaces distributed across the region. Across these 10 surfaces, we identify two distinct populations based on clustering of elevations, which potentially represent remnants of regionally coherent pre-glacial surfaces underlying the West Antarctic Ice Sheet (WAIS). The surfaces are bounded by deeply incised glacial troughs, some of which have potential tectonic controls. We assess two hypotheses for the evolution of the regional landscape: (1) passive-margin evolution associated with the break-up of the Gondwana supercontinent or (2) an extensive planation surface that may have been uplifted in association with either the West Antarctic Rift System or cessation of subduction at the base of the Antarctic Peninsula. We suggest that passive-margin evolution is the most likely of these two mechanisms, with the erosion of glacial troughs adjacent to, and incising, the flat surfaces likely having coincided with the growth of the WAIS. These flat surfaces also demonstrate similarities to other identified surfaces, indicating that a similar formational process may have been acting more widely around the Weddell Sea embayment. The subsequent fluctuations of ice flow, basal thermal regime, and erosion patterns of the WAIS are therefore controlled by the regional tectonic structures

Towards standard setting for patient-reported outcomes in the NHS homeopathic hospitals

We report findings from a pilot data collection study within a programme of quality assurance, improvement and development across all five homeopathic hospitals in the UK National Health Service (NHS).<p></p> <b>Aims</b> (1) To pilot the collection of clinical data in the homeopathic hospital outpatient setting, recording patient-reported outcome since first appointment; (2) to sample the range of medical complaints that secondary-care doctors treat using homeopathy, and thus identify the nature and complexity of complaints most frequently treated nationally; (3) to present a cross section of outcome scores by appointment number, including that for the most frequently treated medical complaints; (4) to explore approaches to standard setting for homeopathic practice outcome in patients treated at the homeopathic hospitals.<p></p> <b>Methods</b> A total of 51 medical practitioners took part in data collection over a 4-week period. Consecutive patient appointments were recorded under the headings: (1) date of first appointment in the current series; (2) appointment number; (3) age of patient; (4) sex of patient; (5) main medical complaint being treated; (6) whether other main medical complaint(s); (7) patient-reported change in health, using Outcome Related to Impact on Daily Living (ORIDL) and its derivative, the ORIDL Profile Score (ORIDL-PS; range, –4 to +4, where a score ≤−2 or ≥+2 indicates an effect on the quality of a patient's daily life); (8) receipt of other complementary medicine for their main medical complaint.<p></p> <b>Results</b> The distribution of patient age was bimodal: main peak, 49 years; secondary peak, 6 years. Male:female ratio was 1:3.5. Data were recorded on a total of 1797 individual patients: 195 first appointments, 1602 follow-ups (FUs). Size of clinical service and proportion of patients who attended more than six visits varied between hospitals. A total of 235 different medical complaints were reported. The 30 most commonly treated complaints were (in decreasing order of frequency): eczema; chronic fatigue syndrome (CFS); menopausal disorder; osteoarthritis; depression; breast cancer; rheumatoid arthritis; asthma; anxiety; irritable bowel syndrome; multiple sclerosis; psoriasis; allergy (unspecified); fibromyalgia; migraine; premenstrual syndrome; chronic rhinitis; headache; vitiligo; seasonal allergic rhinitis; chronic intractable pain; insomnia; ulcerative colitis; acne; psoriatic arthropathy; urticaria; ovarian cancer; attention-deficit hyperactivity disorder (ADHD); epilepsy; sinusitis. The proportion of patients with important co-morbidity was higher in those seen after visit 6 (56.9%) compared with those seen up to and including that point (40.7%; P < 0.001). The proportion of FU patients reporting ORIDL-PS ≥ +2 (improvement affecting daily living) increased overall with appointment number: 34.5% of patients at visit 2 and 59.3% of patients at visit 6, for example. Amongst the four most frequently treated complaints, the proportion of patients that reported ORIDL-PS ≥ +2 at visit numbers greater than 6 varied between 59.3% (CFS) and 73.3% (menopausal disorder).<p></p> <b>Conclusions</b> We have successfully piloted a process of national clinical data collection using patient-reported outcome in homeopathic hospital outpatients, identifying a wide range and complexity of medical complaints treated in that setting. After a series of homeopathy appointments, a high proportion of patients, often representing “effectiveness gaps” for conventional medical treatment, reported improvement in health affecting their daily living. These pilot findings are informing our developing programme of standard setting for homeopathic care in the hospital outpatient context

The Distribution and Origins of Ancient Leprosy

Human leprosy is primarily caused by Mycobacterium leprae, but also by the related ‘M. lepromatosis’. Ancient leprosy can be recognised in archaeological materials by the paleopathology associated with multi-bacillary or lepromatous forms of the disease. Whole M. leprae genomes have been obtained from human skeletons, and diagnostic aDNA fragments have been recovered. The derived M. leprae phylogenies, based on single nucleotide polymorphisms, mirror past human migrations, as M. leprae is usually an obligate pathogen. The detection of M. leprae in historical leprosy cases is assisted by the hydrophobic M. leprae cell envelope, which is composed of unusual lipids that can be used as specific biomarkers. Lipid biomarkers are more stable than aDNA and can be detected directly without amplification. Indigenous human leprosy is extinct in Western Europe, but recently, both M. leprae and ‘M. lepromatosis’ were found in British red squirrels. Leprosy may also be found in nine-banded armadillos (Dasypus novemcinctus) where it can cause a zoonotic human infection. Certain leprosy-like diseases, caused by uncultivable species in cats, for example, may be related to M. leprae. The closest extant relatives of leprosy bacilli are probably members of the M. haemophilum taxon, emerging pathogens with genomic and lipid biomarker similarities

Nutritional Ketosis Alters Fuel Preference and Thereby Endurance Performance in Athletes.

Ketosis, the metabolic response to energy crisis, is a mechanism to sustain life by altering oxidative fuel selection. Often overlooked for its metabolic potential, ketosis is poorly understood outside of starvation or diabetic crisis. Thus, we studied the biochemical advantages of ketosis in humans using a ketone ester-based form of nutrition without the unwanted milieu of endogenous ketone body production by caloric or carbohydrate restriction. In five separate studies of 39 high-performance athletes, we show how this unique metabolic state improves physical endurance by altering fuel competition for oxidative respiration. Ketosis decreased muscle glycolysis and plasma lactate concentrations, while providing an alternative substrate for oxidative phosphorylation. Ketosis increased intramuscular triacylglycerol oxidation during exercise, even in the presence of normal muscle glycogen, co-ingested carbohydrate and elevated insulin. These findings may hold clues to greater human potential and a better understanding of fuel metabolism in health and disease