Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance

Class A serine β-lactamases (SBLs) are key antibiotic resistance determinants in Gram-negative bacteria. SBLs neutralize β-lactams via a hydrolytically labile covalent acyl-enzyme intermediate. Klebsiella pneumoniae carbapenemase (KPC) is a widespread SBL that hydrolyzes carbapenems, the most potent β-lactams; known KPC variants differ in turnover of expanded-spectrum oxyimino-cephalosporins (ESOCs), for example, cefotaxime and ceftazidime. Here, we compare ESOC hydrolysis by the parent enzyme KPC-2 and its clinically observed double variant (P104R/V240G) KPC-4. Kinetic analyses show that KPC-2 hydrolyzes cefotaxime more efficiently than the bulkier ceftazidime, with improved ESOC turnover by KPC-4 resulting from enhanced turnover (k<sub>cat</sub>), rather than altered K<sub>M</sub> values. High-resolution crystal structures of ESOC acyl-enzyme complexes with deacylation-deficient (E166Q) KPC-2 and KPC-4 mutants show that ceftazidime acylation causes rearrangement of three loops; the Ω, 240, and 270 loops, which border the active site. However, these rearrangements are less pronounced in the KPC-4 than the KPC-2 ceftazidime acyl-enzyme and are not observed in the KPC-2:cefotaxime acyl-enzyme. Molecular dynamics simulations of KPC:ceftazidime acyl-enyzmes reveal that the deacylation general base E166, located on the Ω loop, adopts two distinct conformations in KPC-2, either pointing "in" or "out" of the active site; with only the "in" form compatible with deacylation. The "out" conformation was not sampled in the KPC-4 acyl-enzyme, indicating that efficient ESOC breakdown is dependent upon the ordering and conformation of the KPC Ω loop. The results explain how point mutations expand the activity spectrum of the clinically important KPC SBLs to include ESOCs through their effects on the conformational dynamics of the acyl-enzyme intermediate

Recent history, current status, conservation and management of native mammalian carnivore species in Great Britain

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this record After historical declines in population sizes and ranges, we compare and contrast the recent history and contemporary variation in the status of Great Britain's eight native mammalian carnivore species from the 1960s to 2017. Wildcat Felis silvestris conservation status is unfavourable and is masked by hybridisation with domestic cats Felis catus. Red foxes Vulpes vulpes remain widespread but are currently declining. European otter Lutra lutra, European pine marten Martes martes and European polecat Mustela putorius populations are characterised by rapid recovery. Otters have almost completely recolonised Great Britain, polecats have expanded their range throughout southern Britain from refugia in Wales and pine martens have expanded their range from the Scottish Highlands. European badgers Meles meles have generally increased in population density. Status assessments of stoats Mustela erminea and weasels Mustela nivalis are data-deficient but available evidence suggests that stoats may have increased while weasels may have declined. Anthropogenic processes influencing carnivore status include legal protections, habitat quality, reintroductions, predator control, pollutants, hybridisation and diseases and their associated control practices. Population effects of contaminants, such as anticoagulant rodenticides, remain poorly characterised. The widespread interface with domestic and feral cats makes the wildcat's situation precarious. Recent declines in rabbit Oryctolagus cuniculus populations are a concern, given that several carnivore species depend on them as food. We conclude that, with the exception of the wildcat, the status of Great Britain's mammalian carnivores has markedly improved since the 1960s. Better understanding of the social aspects of interactions between humans and expanding predator populations is needed if conflict is to be avoided and long-term co-existence with people is to be possible.Vincent Wildlife TrustUniversity of Exete

Photon indistinguishability measurements under pulsed and continuous excitation

The indistinguishability of successively generated photons from a single quantum emitter is most commonly measured using two-photon interference at a beam splitter. Whilst for sources excited in the pulsed regime the measured bunching of photons reflects the full wavepacket indistinguishability of the emitted photons, for continuous wave (cw) excitation the inevitable dependence on detector timing resolution and driving strength obscures the underlying photon interference process. Here we derive a method to extract the photon indistinguishability from cw measurements by considering the relevant correlation functions. The equivalence of both methods is experimentally verified through comparison of cw and pulsed excitation of an archetypal source of photons, a single molecule

Reduced sensitivity for visual textures affects judgments of shape-from-shading and step climbing behaviour in older adults

Falls on stairs are a major hazard for older adults. Visual decline in normal aging can affect step climbing ability, altering gait and reducing toe clearance. Here we show that a loss of fine-grained visual information associated with age can affect the perception of surface undulations in patterned surfaces. We go on to show that such cues affect the limb trajectories of young adults, but due to their lack of sensitivity, not that of older adults. Interestingly neither the perceived height of a step nor conscious awareness are altered by our visual manipulation but stepping behaviour is: suggesting that the influence of shape perception on stepping behaviour is via the unconscious, action-centred, dorsal visual pathway

Pain assessment for people with dementia: a systematic review of systematic reviews of pain assessment tools.

BACKGROUND: There is evidence of under-detection and poor management of pain in patients with dementia, in both long-term and acute care. Accurate assessment of pain in people with dementia is challenging and pain assessment tools have received considerable attention over the years, with an increasing number of tools made available. Systematic reviews on the evidence of their validity and utility mostly compare different sets of tools. This review of systematic reviews analyses and summarises evidence concerning the psychometric properties and clinical utility of pain assessment tools in adults with dementia or cognitive impairment. METHODS: We searched for systematic reviews of pain assessment tools providing evidence of reliability, validity and clinical utility. Two reviewers independently assessed each review and extracted data from them, with a third reviewer mediating when consensus was not reached. Analysis of the data was carried out collaboratively. The reviews were synthesised using a narrative synthesis approach. RESULTS: We retrieved 441 potentially eligible reviews, 23 met the criteria for inclusion and 8 provided data for extraction. Each review evaluated between 8 and 13 tools, in aggregate providing evidence on a total of 28 tools. The quality of the reviews varied and the reporting often lacked sufficient methodological detail for quality assessment. The 28 tools appear to have been studied in a variety of settings and with varied types of patients. The reviews identified several methodological limitations across the original studies. The lack of a 'gold standard' significantly hinders the evaluation of tools' validity. Most importantly, the samples were small providing limited evidence for use of any of the tools across settings or populations. CONCLUSIONS: There are a considerable number of pain assessment tools available for use with the elderly cognitive impaired population. However there is limited evidence about their reliability, validity and clinical utility. On the basis of this review no one tool can be recommended given the existing evidence

Search algorithms as a framework for the optimization of drug combinations

Combination therapies are often needed for effective clinical outcomes in the management of complex diseases, but presently they are generally based on empirical clinical experience. Here we suggest a novel application of search algorithms, originally developed for digital communication, modified to optimize combinations of therapeutic interventions. In biological experiments measuring the restoration of the decline with age in heart function and exercise capacity in Drosophila melanogaster, we found that search algorithms correctly identified optimal combinations of four drugs with only one third of the tests performed in a fully factorial search. In experiments identifying combinations of three doses of up to six drugs for selective killing of human cancer cells, search algorithms resulted in a highly significant enrichment of selective combinations compared with random searches. In simulations using a network model of cell death, we found that the search algorithms identified the optimal combinations of 6-9 interventions in 80-90% of tests, compared with 15-30% for an equivalent random search. These findings suggest that modified search algorithms from information theory have the potential to enhance the discovery of novel therapeutic drug combinations. This report also helps to frame a biomedical problem that will benefit from an interdisciplinary effort and suggests a general strategy for its solution.Comment: 36 pages, 10 figures, revised versio

The Sewage Sludge Biochar and Its Composts Influence the Phosphate Sorption in an Alkaline–Calcareous Soil

This study evaluates the potential of sewage sludge, farm manure, rock phosphate, and their composts or biochar as useful materials for phosphorous (P) recovery. An alkaline–calcareous soil was incubated with different compost-based amendments to discern their impact on the P sorption characteristics. The treatments examined consisted of sewage sludge (SS), SS biochar (SS-BC), or composts of SS with rock phosphate (RP) and farm manure (FM) in different ratios, i.e., (i) control, (ii) SS, (iii) SS-BC, (iv) SS50:FM50, (v) SS75:FM25, (vi) SS25:FM25:RP50, (vii) SS50:FM25:RP25, (viii) SS75:FM0:RP25, and (ix) SS50:FM0:RP50. Prior to incubation for 45 days, the composts and biochar treatments combinations were characterized for elemental composition using Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) spectroscopy. The treated soils were analyzed for soil pH, electrical conductivity (EC), Olsen’s P, and P adsorption isotherms, at day 5 and 45 of the incubation. Langmuir and Freundlich’s equations were used to calculate P adsorption. Results showed that there was no significant influence on soil pH for any treatment throughout the experiment. However, soil EC decreased significantly for all treatments after 5 days of incubation; however, this effect was diminished after 45 days of incubation. All the treatments significantly decreased P sorption capacity both at the start (day 5) and end of the incubation (day 45) period compared to control. The treatment combination SS25:FM25:RP50 recovered the highest Olsen-P (79.41 mg kg−1). The other treatment combinations including SS and FM also recovered a greater Olsen’s P compared to the control. However, the sole application of only SS showed lower P recovery than the control soil, which further decreased with increase in incubation time. These findings improve our understanding that the stable pool of bioavailable P within an alkaline calcareous soil can be mobilized by using different organic treatment combinations instead of only SS. However, further in situ investigations may help to valorize the potential of sewage sludge, farm manure, rock phosphate, and their composts or biochar to improve P recovery in alkaline–calcareous.</jats:p

Diets of European polecat Mustela putorius in Great Britain during fifty years of population recovery

This is the final version. Available from Springer via the DOI in this record. Following nineteenth-century declines, polecats Mustela putorius are recolonising Great Britain. Polecat diet relates to two potential risks to recovery. First, rabbits Oryctolagus cuniculus, which are important prey for polecats, have experienced extreme population fluctuations, with near extirpation due to myxomatosis in the 1950s, recovery in 1960s–1990s and declines in 1990s–2010s. Second, polecats are secondarily exposed to anticoagulant rodenticides by eating contaminated rodents, and the frequency of polecat exposure to rodenticides is increasing. We analysed stomach contents from 99 polecats collected in 2012–2016 and compared results with earlier studies. Lagomorphs were the most abundant prey (66% frequency of occurrence, 95% confidence interval 53–74%), followed by other mammals (12%, 4–18%), amphibians (10%, 3–16%) and birds (7%, 1–13%). Diet varied seasonally; lagomorph occurrence was highest in spring and summer and lowest in autumn. Dietary niche breadth was greater in the 1960s, when rabbits were scarce, than in other decades, but did not differ between the 1990s and 2010s, indicating that diets have not diversified with recent rabbit declines. This may be because rabbit abundance is not yet low enough to cause dietary diversification or because polecats were collected in areas where rabbits were still abundant. Rodents did not increase in diet between the 1990s and 2010s and still occur with < 10% frequency, indicating that rodents need not contribute much to diet to expose polecats to rodenticides. This potentially limits the effectiveness of management actions designed to minimise polecat exposure to contaminated rodent prey.Vincent Wildlife TrustUniversity of Exete