Adaptive designs are a class of methods for improving efficiency and patient benefit of clinical trials. Although their use has increased in recent years, research suggests they are not used in many ...situations where they have potential to bring benefit. One barrier to their more widespread use is a lack of understanding about how the choice to use an adaptive design, rather than a traditional design, affects resources (staff and non-staff) required to set-up, conduct and report a trial. The Costing Adaptive Trials project investigated this issue using quantitative and qualitative research amongst UK Clinical Trials Units. Here, we present guidance that is informed by our research, on considering the appropriate resourcing of adaptive trials. We outline a five-step process to estimate the resources required and provide an accompanying costing tool. The process involves understanding the tasks required to undertake a trial, and how the adaptive design affects them. We identify barriers in the publicly funded landscape and provide recommendations to trial funders that would address them. Although our guidance and recommendations are most relevant to UK non-commercial trials, many aspects are relevant more widely.
•Senescent sweetening in potato was associated with significant alterations in transcripts associated with sugar-starch metabolism.•Evidence suggest a plastidial glucose-6-phosphate/phosphate ...translocator (GPT2) plays a significant role in senescent sweetening.•A potential mechanism of senescent sweetening is a reduction in the capacity to import sugars into the amyloplast for starch resynthesis.•GPT2 represents a target for improved senescent sweetening resistance via breeding or biotechnology.
Senescent sweetening results in the accumulation of reducing sugars in potato tubers following extended periods of storage at moderate temperatures used to avoid the separate condition of cold-induced sweetening. It represents a significant problem for the potato processing industry due to the development of dark fry colour and the accumulation of acrylamide in processed products. Previous studies have implicated oxidative stress in the accumulation of reducing sugars in potato tubers over long term storage. However, in the present analysis we found no evidence for a correlation between oxidative stress as estimated from quantification of hydrogen peroxide, lipid peroxidation or activity of redox enzymes and the accumulation of reducing sugars. On the contrary, transcriptional profiling indicated changes in carbohydrate metabolism were associated with the onset of senescent sweetening and qRT-PCR indicated that reduced abundance of transcripts encoding a plastidial glucose-6-phosphate/phosphate translocator was widely observed during sweetening onset in multiple genotypes. Our data suggest that reduction in the capacity of plastids to import glucose-6-phosphate reduces the capacity for starch resynthesis in the stored tuber thereby shifting the metabolic balance towards starch turnover resulting in reducing sugar accumulation.
Incompatibility group C (IncC) plasmids are large (50–400 kb), broad host range plasmids that drive the spread of genes conferring resistance to all classes of antibiotics, most notably the blaNDM ...gene that confers resistance to last‐line carbapenems and the mcr‐3 gene that confers resistance to colistin. Several recent studies have improved our understanding of the basic biological mechanisms driving the success of IncC, in particular the identification of multiple novel IncC conjugation genes by transposon directed insertion‐site sequencing. Here, one of these genes, dtrJ, was examined in further detail. The dtrJ gene is located in the DNA transfer locus on the IncC backbone, and quantitative reverse‐transcriptase PCR analysis revealed it is transcribed in the same operon as the DNA transfer genes traI and traD (encoding the relaxase and coupling protein, respectively) and activated by the AcaDC regulatory complex. We confirmed that DtrJ is not required for pilus biogenesis or mate pair formation. Instead, DtrJ localizes to the membrane, where it interacts with the coupling protein TraD and functions as an IncC DNA transfer protein. Overall, this work has defined the role of DtrJ in DNA transfer of IncC plasmids during conjugation.
This manuscript describes the function of the conjugation protein, DtrJ. We show that DtrJ is critical for the DNA transfer step of plasmid conjugation and may be involved in specific DNA substrate selection during conjugation.
Carbapenem-resistant
are urgent threats to global human health. These organisms produce β-lactamases with carbapenemase activity, such as the metallo-β-lactamase NDM-1, which is notable due to its ...association with mobile genetic elements and the lack of a clinically useful inhibitor. Here we examined the ability of copper to inhibit the activity of NDM-1 and explored the potential of a copper coordination complex as a mechanism to efficiently deliver copper as an adjuvant in clinical therapeutics. An NDM-positive
isolate, MS6192, was cultured from the urine of a patient with a urinary tract infection. MS6192 was resistant to antibiotics from multiple classes, including diverse β-lactams (penicillins, cephalosporins, and carbapenems), aminoglycosides, and fluoroquinolones. In the presence of copper (range, 0 to 2 mM), however, the susceptibility of MS6192 to the carbapenems ertapenem and meropenem increased markedly. In standard checkerboard assays, copper decreased the MICs of ertapenem and meropenem against MS6192 in a dose-dependent manner, suggesting a synergistic mode of action. To examine the inhibitory effect of copper in the absence of other β-lactamases, the
gene from MS6192 was cloned and expressed in a recombinant
K-12 strain. Analysis of cell extracts prepared from this strain revealed that copper directly inhibited NDM-1 activity, which was confirmed using purified recombinant NDM-1. Finally, delivery of copper at a low concentration of 10 μM by using the FDA-approved coordination complex copper-pyrithione sensitized MS6192 to ertapenem and meropenem in a synergistic manner. Overall, this work demonstrates the potential use of copper coordination complexes as novel carbapenemase adjuvants.
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•Ivermectin promotes the polymerisation and stability of Haemonchus contortus microtubules.•Haemonchus contortus α-tubulin and β-tubulin isotype 1 have an ivermectin binding ...site.•Ivermectin and Taxol have overlapping binding sites and compete for binding to H. contortus tubulin.
Haemonchus contortus is a nematode of livestock that can cause severe disease and mortality. Ivermectin, an anti-parasitic drug that targets glutamate-gated chloride channels, is widely used in humans, livestock, companion animals and agriculture. Although an association between genetic changes to β-tubulin and exposure to ivermectin has been previously reported, direct binding between ivermectin and tubulin has not been demonstrated to date. Tubulin/microtubules are key targets for many anti-mitotic drugs used in anti-parasite and cancer therapies. We now report that ivermectin exposure increased the rate and extent of polymerisation of H. contortus recombinant α- and β-tubulin, and protected the parasitic α- and β-tubulins from limited trypsin proteolysis. Direct binding between ivermectin and the tubulin monomers exhibited low micromolar affinities, as determined using surface plasmon resonance. Subsequent equilibrium dialysis indicated that ivermectin and Taxol compete for binding to tubulin, supporting our molecular modelling that predicts ivermectin interacts with the Taxol binding pocket of both parasitic and mammalian tubulins. Collectively, our data indicate that ivermectin can bind to and stabilise microtubules (i.e. alter the tubulin polymerisation equilibrium) and this can then lead to mitotic arrest. This work extends the range of known pharmacological effects of ivermectin, and reveals its potential as an anti-mitotic agent.
Hypertension is a key risk factor for major adverse cardiovascular events but remains difficult to treat in many individuals. Dietary interventions are an effective approach to lower blood pressure ...(BP) but are not equally effective across all individuals. BP is heritable, and genetics may be a useful tool to overcome treatment response heterogeneity. We investigated whether the genetics of BP could be used to identify individuals with hypertension who may receive a particular benefit from lowering sodium intake and boosting potassium levels.
In this observational genetic study, we leveraged cross-sectional data from up to 296 475 genotyped individuals drawn from the UK Biobank cohort for whom BP and urinary electrolytes (sodium and potassium), biomarkers of sodium and potassium intake, were measured. Biologically directed genetic scores for BP were constructed specifically among pathways related to sodium and potassium biology (pharmagenic enrichment scores), as well as unannotated genome-wide scores (conventional polygenic scores). We then tested whether there was a gene-by-environment interaction between urinary electrolytes and these genetic scores on BP.
Genetic risk and urinary electrolytes both independently correlated with BP. However, urinary sodium was associated with a larger BP increase among individuals with higher genetic risk in sodium- and potassium-related pathways than in those with comparatively lower genetic risk. For example, each SD in urinary sodium was associated with a 1.47-mm Hg increase in systolic BP for those in the top 10% of the distribution of genetic risk in sodium and potassium transport pathways versus a 0.97-mm Hg systolic BP increase in the lowest 10% (
=1.95×10
). This interaction with urinary sodium remained when considering estimated glomerular filtration rate and indexing sodium to urinary creatinine. There was no strong evidence of an interaction between urinary sodium and a standard genome-wide polygenic score of BP.
The data suggest that genetic risk in sodium and potassium pathways could be used in a precision medicine model to direct interventions more specifically in the management of hypertension. Intervention studies are warranted.
Adaptive designs offer great promise in improving the efficiency and patient-benefit of clinical trials. An important barrier to further increased use is a lack of understanding about which ...additional resources are required to conduct a high-quality adaptive clinical trial, compared to a traditional fixed design. The Costing Adaptive Trials (CAT) project investigated which additional resources may be required to support adaptive trials.
We conducted a mock costing exercise amongst seven Clinical Trials Units (CTUs) in the UK. Five scenarios were developed, derived from funded clinical trials, where a non-adaptive version and an adaptive version were described. Each scenario represented a different type of adaptive design. CTU staff were asked to provide the costs and staff time they estimated would be needed to support the trial, categorised into specified areas (e.g. statistics, data management, trial management). This was calculated separately for the non-adaptive and adaptive version of the trial, allowing paired comparisons. Interviews with 10 CTU staff who had completed the costing exercise were conducted by qualitative researchers to explore reasons for similarities and differences.
Estimated resources associated with conducting an adaptive trial were always (moderately) higher than for the non-adaptive equivalent. The median increase was between 2 and 4% for all scenarios, except for sample size re-estimation which was 26.5% (as the adaptive design could lead to a lengthened study period). The highest increase was for statistical staff, with lower increases for data management and trial management staff. The percentage increase in resources varied across different CTUs. The interviews identified possible explanations for differences, including (1) experience in adaptive trials, (2) the complexity of the non-adaptive and adaptive design, and (3) the extent of non-trial specific core infrastructure funding the CTU had.
This work sheds light on additional resources required to adequately support a high-quality adaptive trial. The percentage increase in costs for supporting an adaptive trial was generally modest and should not be a barrier to adaptive designs being cost-effective to use in practice. Informed by the results of this research, guidance for investigators and funders will be developed on appropriately resourcing adaptive trials.
A series of three platinum(II) phenanthroimidazoles each containing a protonable side‐chain appended from the phenyl moiety through copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) were ...evaluated for their capacities to bind to human telomere, c‐Myc, and c‐Kit derived G‐quadruplexes. The side‐chain has been optimized to enable a multivalent binding mode to G‐quadruplex motifs, which would potentially result in selective targeting. Molecular modeling, high‐throughput fluorescence intercalator displacement (HT‐FID) assays, and surface plasmon resonance (SPR) studies demonstrate that complex 2 exhibits significantly slower dissociation rates compared to platinum phenanthroimidazoles without side‐chains and other reported G‐quadruplex binders. Complex 2 showed little cytotoxicity in HeLa and A172 cancer cell lines, consistent with the fact that it does not follow a telomere‐targeting pathway. Preliminary mRNA analysis shows that 2 specifically interacts with the ckit promoter region. Overall, this study validates 2 as a useful molecular probe for c‐Kit related cancer pathways.
Bound to impress: A series of three platinum(II) phenanthroimidazoles each containing a protonable side‐chain appended from the phenyl moiety through copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC), was evaluated for their capacity to bind to human telomere, c‐Myc, and c‐Kit derived G‐quadruplexes (see scheme). The presence of the side‐chain enables a multivalent binding mode to G‐quadruplex motifs.
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