TraDIS-Xpress: a high-resolution whole-genome assay identifies novel mechanisms of triclosan action and resistance.

Understanding the genetic basis for a phenotype is a central goal in biological research. Much has been learnt about bacterial genomes by creating large mutant libraries and looking for conditionally important genes. However, current genome-wide methods are largely unable to assay essential genes which are not amenable to disruption. To overcome this limitation, we developed a new version of "TraDIS" (transposon directed insertion-site sequencing) that we term "TraDIS-Xpress" that combines an inducible promoter into the transposon cassette. This allows controlled overexpression and repression of all genes owing to saturation of inserts adjacent to all open reading frames as well as conventional inactivation. We applied TraDIS-Xpress to identify responses to the biocide triclosan across a range of concentrations. Triclosan is endemic in modern life, but there is uncertainty about its mode of action with a concentration-dependent switch from bacteriostatic to bactericidal action unexplained. Our results show a concentration-dependent response to triclosan with different genes important in survival between static and cidal exposures. These genes include those previously reported to have a role in triclosan resistance as well as a new set of genes, including essential genes. Novel genes identified as being sensitive to triclosan exposure include those involved in barrier function, small molecule uptake, and integrity of transcription and translation. We anticipate the approach we show here, by allowing comparisons across multiple experimental conditions of TraDIS data, and including essential genes, will be a starting point for future work examining how different drug conditions impact bacterial survival mechanisms.

Investigating Antibody Reactivity to the Intestinal Microbiome in Severe Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS): A Feasibility Study.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multisystemic disease of unknown aetiology that is characterised by disabling chronic fatigue and involves both the immune and gastrointestinal (GI) systems. Patients display alterations in GI microbiome with a significant proportion experiencing GI discomfort and pain and elevated blood biomarkers for altered intestinal permeability compared with healthy individuals. To investigate a possible GI origin of ME/CFS we designed a feasibility study to test the hypothesis that ME/CFS pathogenesis is a consequence of increased intestinal permeability that results in microbial translocation and a breakdown in immune tolerance leading to generation of antibodies reactive to indigenous intestinal microbes. Secretory immunoglobulin (Ig) A and serum IgG levels and reactivity to intestinal microbes were assessed in five pairs of severe ME/CFS patients and matched same-household healthy controls. For profiling serum IgG, we developed IgG-Seq which combines flow-cytometry based bacterial cell sorting and metagenomics to detect mucosal IgG reactivity to the microbiome. We uncovered evidence for immune dysfunction in severe ME/CFS patients that was characterised by reduced capacity and reactivity of serum IgG to stool microbes, irrespective of their source. This study provides the rationale for additional studies in larger cohorts of ME/CFS patients to further explore immune-microbiome interactions.

Investigating the Human Intestinal DNA Virome and Predicting Disease-Associated Virus-Host Interactions in Severe Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

Understanding how the human virome, and which of its constituents, contributes to health or disease states is reliant on obtaining comprehensive virome profiles. By combining DNA viromes from isolated virus-like particles (VLPs) and whole metagenomes from the same faecal sample of a small cohort of healthy individuals and patients with severe myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), we have obtained a more inclusive profile of the human intestinal DNA virome. Key features are the identification of a core virome comprising tailed phages of the class Caudoviricetes, and a greater diversity of DNA viruses including extracellular phages and integrated prophages. Using an in silico approach, we predicted interactions between members of the Anaerotruncus genus and unique viruses present in ME/CFS microbiomes. This study therefore provides a framework and rationale for studies of larger cohorts of patients to further investigate disease-associated interactions between the intestinal virome and the bacteriome.

Multiple holins contribute to extracellular DNA release in Pseudomonas aeruginosa biofilms.

Bacterial biofilms are composed of aggregates of cells encased within a matrix of extracellular polymeric substances (EPS). One key EPS component is extracellular DNA (eDNA), which acts as a 'glue', facilitating cell-cell and cell-substratum interactions. We have previously demonstrated that eDNA is produced in Pseudomonas aeruginosa biofilms via explosive cell lysis. This phenomenon involves a subset of the bacterial population explosively lysing, due to peptidoglycan degradation by the endolysin Lys. Here we demonstrate that in P. aeruginosa three holins, AlpB, CidA and Hol, are involved in Lys-mediated eDNA release within both submerged (hydrated) and interstitial (actively expanding) biofilms, albeit to different extents, depending upon the type of biofilm and the stage of biofilm development. We also demonstrate that eDNA release events determine the sites at which cells begin to cluster to initiate microcolony formation during the early stages of submerged biofilm development. Furthermore, our results show that sustained release of eDNA is required for cell cluster consolidation and subsequent microcolony development in submerged biofilms. Overall, this study adds to our understanding of how eDNA release is controlled temporally and spatially within P. aeruginosa biofilms.

AlbaTraDIS: Comparative analysis of large datasets from parallel transposon mutagenesis experiments.

Bacteria need to survive in a wide range of environments. Currently, there is an incomplete understanding of the genetic basis for mechanisms underpinning survival in stressful conditions, such as the presence of anti-microbials. Transposon directed insertion-site sequencing (TraDIS) is a powerful tool to identify genes and networks which are involved in survival and fitness under a given condition by simultaneously assaying the fitness of millions of mutants, thereby relating genotype to phenotype and contributing to an understanding of bacterial cell biology. A recent refinement of this approach allows the roles of essential genes in conditional stress survival to be inferred by altering their expression. These advancements combined with the rapidly falling costs of sequencing now allows comparisons between multiple experiments to identify commonalities in stress responses to different conditions. This capacity however poses a new challenge for analysis of multiple data sets in conjunction. To address this analysis need, we have developed 'AlbaTraDIS'; a software application for rapid large-scale comparative analysis of TraDIS experiments that predicts the impact of transposon insertions on nearby genes. AlbaTraDIS can identify genes which are up or down regulated, or inactivated, between multiple conditions, producing a filtered list of genes for further experimental validation as well as several accompanying data visualisations. We demonstrate the utility of our new approach by applying it to identify genes used by Escherichia coli to survive in a wide range of different concentrations of the biocide Triclosan. AlbaTraDIS identified all well characterised Triclosan resistance genes, including the primary target, fabI. A number of new loci were also implicated in Triclosan resistance and the predicted phenotypes for a selection of these were validated experimentally with results being consistent with predictions. AlbaTraDIS provides a simple and rapid method to analyse multiple transposon mutagenesis data sets allowing this technology to be used at large scale. To our knowledge this is the only tool currently available that can perform these tasks. AlbaTraDIS is written in Python 3 and is available under the open source licence GNU GPL 3 from https://github.com/quadram-institute-bioscience/albatradis.

Cognitive decline associated with anticholinergics, benzodiazepines and Z-drugs: Findings from The Irish Longitudinal Study on Ageing (TILDA).

AIMS: To estimate the association between patterns of anticholinergic, benzodiazepine and Z-drug medication use and change in cognitive function in middle-aged and older adults. METHODS: This prospective cohort study used data from the first three waves of The Irish Longitudinal Study on Ageing (TILDA), including community-dwelling adults aged ≥50 years followed for up to 4 years (n = 7027). Cognitive function was assessed using the Mini Mental State Examination, animal naming test and word recall tests. Regular medication use was self-reported at baseline and follow-up interviews at 2 and 4 years. Pharmacy dispensing claims for a subset (n = 2905) allowed assessment of medication use between interviews and cumulative dosage. Medication use at consecutive waves of TILDA was analysed in relation to change in cognitive function between waves. RESULTS: Strongly anticholinergic medications (Anticholinergic Cognitive Burden scale 3), benzodiazepines and Z-drugs were reported by 7.3%, 5.8% and 5.1% of participants, respectively, at any time during the study. Adjusting for potential confounders, new anticholinergic use between interviews was associated with change in recall score (-1.09, 95% confidence interval -1.64, -0.53) over 2 years compared to non-use, but not with MMSE (0.07; 95% CI -0.21, 0.34) or animal naming (-0.70; 95% CI -1.43, 0.03). The pharmacy claims analysis was consistent with this finding. Other hypothesised associations were not supported. CONCLUSIONS: Except for new use of anticholinergic medications, no other findings supported a risk of cognitive decline over 2-year periods in this middle-aged and older cohort. Patients and prescribers should weigh this potential risk against potential benefits of commencing anticholinergic medications.

The use of an in-vitro batch fermentation (human colon) model for investigating mechanisms of TMA production from choline, L-carnitine and related precursors by the human gut microbiota.

PURPOSE: Plasma trimethylamine-N-oxide (TMAO) levels have been shown to correlate with increased risk of metabolic diseases including cardiovascular diseases. TMAO exposure predominantly occurs as a consequence of gut microbiota-dependent trimethylamine (TMA) production from dietary substrates including choline, carnitine and betaine, which is then converted to TMAO in the liver. Reducing microbial TMA production is likely to be the most effective and sustainable approach to overcoming TMAO burden in humans. Current models for studying microbial TMA production have numerous weaknesses including the cost and length of human studies, differences in TMA(O) metabolism in animal models and the risk of failing to replicate multi-enzyme/multi-strain pathways when using isolated bacterial strains. The purpose of this research was to investigate TMA production from dietary precursors in an in-vitro model of the human colon. METHODS: TMA production from choline, L-carnitine, betaine and γ-butyrobetaine was studied over 24-48 h using an in-vitro human colon model with metabolite quantification performed using LC-MS. RESULTS: Choline was metabolised via the direct choline TMA-lyase route but not the indirect choline-betaine-TMA route, conversion of L-carnitine to TMA was slower than that of choline and involves the formation of the intermediate γ-BB, whereas the Rieske-type monooxygenase/reductase pathway for L-carnitine metabolism to TMA was negligible. The rate of TMA production from precursors was choline > carnitine > betaine > γ-BB. 3,3-Dimethyl-1-butanol (DMB) had no effect on the conversion of choline to TMA. CONCLUSION: The metabolic routes for microbial TMA production in the colon model are consistent with observations from human studies. Thus, this model is suitable for studying gut microbiota metabolism of TMA and for screening potential therapeutic targets that aim to attenuate TMA production by the gut microbiota. TRIAL REGISTRATION NUMBER: NCT02653001 ( http://www.clinicaltrials.gov ), registered 12 Jan 2016.

The Pregnancy and EARly Life study (PEARL) - a longitudinal study to understand how gut microbes contribute to maintaining health during pregnancy and early life.

BACKGROUND: The early life period represents the first step in establishing a beneficial microbial ecosystem, which in turn affects both short and longer-term health. Changes during pregnancy influence the neonatal microbiome; through transmission of maternal microbes during childbirth, and beyond, through nutritional programming. However, in-depth exploration of longitudinal maternal-infant cohorts, with sampling of multiple body sites, complemented by clinical and nutritional metadata, and use of cutting-edge experimental systems are limited. The PEARL study will increase our knowledge of; how microbes (including viruses/phages, bacteria, fungi and archaea) change in composition and functional capacity during pregnancy; transmission pathways from mother to infant; the impact of various factors on microbial communities across pregnancy and early life (e.g. diet), and how these microbes interact with other microbes and modulate host processes, including links to disease onset. METHODS: PEARL is a longitudinal observational prospective study of 250 pregnant women and their newborns, with stool and blood samples, questionnaires and routine clinical data collected during pregnancy, labour, birth and up to 24 months post birth. Metagenomic sequencing of samples will be used to define microbiome profiles, and allow for genus, species and strain-level taxonomic identification and corresponding functional analysis. A subset of samples will be analysed for host (immune/metabolite) molecules to identify factors that alter the host gut environment. Culturing will be used to identify new strains of health-promoting bacteria, and potential pathogens. Various in vitro and in vivo experiments will probe underlying mechanisms governing microbe-microbe and microbe-host interactions. DISCUSSION: Longitudinal studies, like PEARL, are critical if we are to define biomarkers, determine mechanisms underlying microbiome profiles in health and disease, and develop new diet- and microbe-based therapies to be tested in future studies and clinical trials. TRIAL REGISTRATION: This study is registered in the ClinicalTrials.gov Database with ID: NCT03916874 .

Adverse effects of Z-drugs for sleep disturbance in people living with dementia: a population-based cohort study.

BACKGROUND: Sleep disturbance is common in dementia and often treated with Z-drugs (zopiclone, zaleplon, and zolpidem). While some observational studies suggest that Z-drugs are associated with adverse events such as falls and fracture risks in older people, this has not been studied in dementia. METHODS: We used data from 27,090 patients diagnosed with dementia between January 2000 and March 2016 from the Clinical Practice Research Datalink linked to Hospital Episodes Statistics data in England. We compared adverse events for 3532 patients newly prescribed Z-drugs by time-varying dosage to (1) 1833 non-sedative-users with sleep disturbance; (2) 10,214 non-sedative-users with proximal GP consultation matched on age, sex, and antipsychotic use; and (3) 5172 patients newly prescribed benzodiazepines. We defined higher dose Z-drugs and benzodiazepines as prescriptions equivalent to ≥ 7.5 mg zopiclone or > 5 mg diazepam daily. Cox regression was used to estimate hazard ratios (HRs) for incident fracture, hip fracture, fall, mortality, acute bacterial infection, ischaemic stroke/transient ischaemic attack, and venous thromboembolism over a 2-year follow-up, adjusted for demographic- and health-related covariates. RESULTS: The mean (SD) age of patients was 83 (7.7) years, and 16,802 (62%) were women. Of 3532 patients prescribed Z-drugs, 584 (17%) were initiated at higher doses. For patients prescribed higher dose Z-drugs relative to non-users with sleep disturbance, the HRs (95% confidence interval) for fractures, hip fractures, falls, and ischaemic stroke were 1.67 (1.13-2.46), 1.96 (1.16-3.31), 1.33 (1.06-1.66), and 1.88 (1.14-3.10), respectively. We observed similar associations when compared to non-sedative-users with proximal GP consultation. Minimal or inconsistent excess risks were observed at ≤ 3.75 mg zopiclone or equivalent daily, and for mortality, infection, and venous thromboembolism. We observed no differences in adverse events for Z-drugs compared to benzodiazepines, except lower mortality rates with Z-drugs (HR [95% confidence interval] of 0.73 [0.64-0.83]). CONCLUSIONS: Higher dose Z-drug use in dementia is associated with increased fracture and stroke risks, similar or greater to that for higher dose benzodiazepines. Higher dose Z-drugs should be avoided, if possible, in people living with dementia, and non-pharmacological alternatives preferentially considered. Prescriptions for higher dose Z-drugs in dementia should be regularly reviewed. TRIAL REGISTRATION: ENCePP e-register of studies, EUPAS18006.

Changing prevalence and treatment of depression among older people over two decades.

BACKGROUND: Depression is a leading cause of disability, with older people particularly susceptible to poor outcomes. AIMS: To investigate whether the prevalence of depression and antidepressant use have changed across two decades in older people. METHOD: The Cognitive Function and Ageing Studies (CFAS I and CFAS II) are two English population-based cohort studies of older people aged ≥65 years, with baseline measurements for each cohort conducted two decades apart (between 1990 and 1993 and between 2008 and 2011). Depression was assessed by the Geriatric Mental State examination and diagnosed with the Automated Geriatric Examination for Computer-Assisted Taxonomy algorithm. RESULTS: In CFAS I, 7635 people aged ≥65 years were interviewed, of whom 1457 were diagnostically assessed. In CFAS II, 7762 people were interviewed and diagnostically assessed. Age-standardised depression prevalence in CFAS II was 6.8% (95% CI 6.3-7.5%), representing a non-significant decline from CFAS I (risk ratio 0.82, 95% CI 0.64-1.07, P = 0.14). At the time of CFAS II, 10.7% of the population (95% CI 10.0-11.5%) were taking antidepressant medication, more than twice that of CFAS I (risk ratio 2.79, 95% CI 1.96-3.97, P < 0.0001). Among care home residents, depression prevalence was unchanged, but the use of antidepressants increased from 7.4% (95% CI 3.8-13.8%) to 29.2% (95% CI 22.6-36.7%). CONCLUSIONS: A substantial increase in the proportion of the population reporting taking antidepressant medication is seen across two decades for people aged ≥65 years. However there was no evidence for a change in age-specific prevalence of depression.

Anticholinergic drugs and incident dementia, mild cognitive impairment and cognitive decline: a meta-analysis.

BACKGROUND: the long-term effect of the use of drugs with anticholinergic activity on cognitive function remains unclear. METHODS: we conducted a systematic review and meta-analysis of the relationship between anticholinergic drugs and risk of dementia, mild cognitive impairment (MCI) and cognitive decline in the older population. We identified studies published between January 2002 and April 2018 with ≥12 weeks follow-up between strongly anticholinergic drug exposure and the study outcome measurement. We pooled adjusted odds ratios (OR) for studies reporting any, and at least short-term (90+ days) or long-term (365+ days) anticholinergic use for dementia and MCI outcomes, and standardised mean differences (SMD) in global cognition test scores for cognitive decline outcomes. Statistical heterogeneity was measured using the I2 statistic and risk of bias using ROBINS-I. RESULTS: twenty-six studies (including 621,548 participants) met our inclusion criteria. 'Any' anticholinergic use was associated with incident dementia (OR 1.20, 95% confidence interval [CI] 1.09-1.32, I2 = 86%). Short-term and long-term use were also associated with incident dementia (OR 1.23, 95% CI 1.17-1.29, I2 = 2%; and OR 1.50, 95% CI 1.22-1.85, I2 = 90%). 'Any' anticholinergic use was associated with cognitive decline (SMD 0.15; 95% CI 0.09-0.21, I2 = 3%) but showed no statistically significant difference for MCI (OR 1.24, 95% CI 0.97-1.59, I2 = 0%). CONCLUSIONS: anticholinergic drug use is associated with increased dementia incidence and cognitive decline in observational studies. However, a causal link cannot yet be inferred, as studies were observational with considerable risk of bias. Stronger evidence from high-quality studies is needed to guide the management of long-term use.

Increasing prevalence of anticholinergic medication use in older people in England over 20 years: cognitive function and ageing study I and II.

BACKGROUND: Anticholinergic medication use is linked with increased cognitive decline, dementia, falls and mortality, and their use should be limited in older people. Here we estimate the prevalence of anticholinergic use in England's older population in 1991 and 2011, and describe changes in use by participant's age, sex, cognition and disability. METHODS: We compared data from participants aged 65+ years from the Cognitive Function and Ageing Studies (CFAS I and II), collected during 1990-1993 (N = 7635) and 2008-2011 (N = 7762). We estimated the prevalence of potent anticholinergic use (Anticholinergic Cognitive Burden [ACB] score = 3) and average anticholinergic burden (sum of ACB scores), using inverse probability weights standardised to the 2011 UK population. These were stratified by age, sex, Mini-Mental State Examination score, and activities of daily living (ADL) or instrumental ADL (IADL) disability. RESULTS: Prevalence of potent anticholinergic use increased from 5.7% (95% Confidence Interval [CI] 5.2-6.3%) of the older population in 1990-93 to 9.9% (9.3-10.7%) in 2008-11, adjusted odds ratio of 1.90 (95% CI 1.67-2.16). People with clinically significant cognitive impairment (MMSE [Mini Mental State Examination] 21 or less) were the heaviest users of potent anticholinergics in CFAS II (16.5% [95% CI 12.0-22.3%]). Large increases in the prevalence of the use medication with 'any' anticholinergic activity were seen in older people with clinically significant cognitive impairment (53.3% in CFAS I to 71.5% in CFAS II). CONCLUSIONS: Use of potent anticholinergic medications nearly doubled in England's older population over 20 years with some of the greatest increases amongst those particularly vulnerable to anticholinergic side-effects.

History of Benzodiazepine Prescriptions and Risk of Dementia: Possible Bias Due to Prevalent Users and Covariate Measurement Timing in a Nested Case-Control Study.

Previous estimates of whether long-term exposure to benzodiazepines increases dementia risk are conflicting and are compromised by the difficulty of controlling for confounders and by reverse causation. We investigated how estimates for the association between benzodiazepine use and later dementia incidence varied based on study design choices, using a case-control study nested within the United Kingdom's Clinical Practice Research Datalink. A total of 40,770 dementia cases diagnosed between April 2006 and July 2015 were matched on age, sex, available data history, and deprivation to 283,933 control subjects. Benzodiazepines and Z-drug prescriptions were ascertained in a drug-exposure period 4-20 years before dementia diagnosis. Estimates varied with the inclusion of new or prevalent users, with the timing of covariate ascertainment, and with varying time between exposure and outcome. There was no association between any new prescription of benzodiazepines and dementia (adjusted odds ratio (OR) = 1.03, 95% confidence interval (CI): 1.00, 1.07), whereas an inverse association was observed among prevalent users (adjusted OR = 0.91, 95% CI: 0.87, 0.95), although this was likely induced by unintentional adjustment for colliders. By considering the choice of confounders and timing of exposure and covariate measurement, our findings overall are consistent with no causal effect of benzodiazepines or Z-drugs on dementia incidence.

The effects of hypoglycaemia and dementia on cardiovascular events, falls and fractures and all-cause mortality in older individuals: A retrospective cohort study.

AIMS: Older individuals with diabetes are susceptible to harm as the result of hypoglycaemia; however, the consequences of hypoglycaemia in older individuals with dementia are not known. We aimed to test the association between hypoglycaemia and serious adverse events in older patients with diabetes and dementia, and whether the consequences of hypoglycaemia were affected by the presence of dementia. MATERIALS AND METHODS: This was a cohort study using the Clinical Practice Research Datalink in England (1997-2016). We selected participants, intervention (exposure) and follow-up to mirror two hypothetical target randomized controlled trials. The exposure of target trial 1 was hypoglycaemia in patients with dementia. Target trial 2 examined adverse effects of hypoglycaemia according to dementia status. We used Cox proportional hazard regression to estimate adjusted hazard ratios (aHR) for falls, fractures, cardiovascular events and mortality. RESULTS: In target trial 1, hypoglycaemia was associated with increased risk during a 12-month follow-up period for falls and fractures (aHR, 1.94 [95% CI, 1.67-2.24]), for cardiovascular events (aHR, 2.00 [95% CI, 1.61-2.48]) and for mortality (aHR, 2.36 [95% CI, 2.09-2.67]). In target trial 2, the presence of dementia was associated with increased risk of adverse events, following hypoglycaemia, during a 12-month follow-up period for falls and factures (aHR, 1.72 [95% CI, 1.51-1.96]) and for mortality (aHR, 1.27 [95% CI, 1.15-1.41]), but dementia had no effect on cardiovascular events (aHR, 1.14 [95% CI, 0.95 to 1.36]). CONCLUSIONS: Hypoglycaemia is associated with early increased risk of serious adverse events in older individuals with diabetes and dementia.

Anticholinergic and benzodiazepine medication use and risk of incident dementia: a UK cohort study.

BACKGROUND: Studies suggest that anticholinergic medication or benzodiazepine use could increase dementia risk. We tested this hypothesis using data from a UK cohort study. METHODS: We used data from the baseline (Y0), 2-year (Y2) and 10-year (Y10) waves of the Medical Research Council Cognitive Function and Ageing Study. Participants without dementia at Y2 were included (n = 8216). Use of benzodiazepines (including nonbenzodiazepine Z-drugs), anticholinergics with score 3 (ACB3) and anticholinergics with score 1 or 2 (ACB12) according to the Anticholinergic Cognitive Burden scale were coded as ever use (use at Y0 or Y2), recurrent use (Y0 and Y2), new use (Y2, but not Y0) or discontinued use (Y0, but not Y2). The outcome was incident dementia by Y10. Incidence rate ratios (IRR) were estimated using Poisson regression adjusted for potential confounders. Pre-planned subgroup analyses were conducted by age, sex and Y2 Mini-Mental State Examination (MMSE) score. RESULTS: Dementia incidence was 9.3% (N = 220 cases) between Y2 and Y10. The adjusted IRRs (95%CI) of developing dementia were 1.06 (0.72, 1.60), 1.28 (0.82, 2.00) and 0.89 (0.68, 1.17) for benzodiazepines, ACB3 and ACB12 ever-users compared with non-users. For recurrent users the respective IRRs were 1.30 (0.79, 2.14), 1.68 (1.00, 2.82) and 0.95 (0.71, 1.28). ACB3 ever-use was associated with dementia among those with Y2 MMSE> 25 (IRR = 2.28 [1.32-3.92]), but not if Y2 MMSE≤25 (IRR = 0.94 [0.51-1.73]). CONCLUSIONS: Neither benzodiazepines nor ACB12 medications were associated with dementia. Recurrent use of ACB3 anticholinergics was associated with dementia, particularly in those with good baseline cognitive function. The long-term prescribing of anticholinergics should be avoided in older people.

Is frailty a stable predictor of mortality across time? Evidence from the Cognitive Function and Ageing Studies.

Background: age-specific mortality reduction has been accompanied by a decrease in the prevalence of some diseases and an increase in others. Whether populations are becoming 'healthier' depends on which aspect of health is being considered. Frailty has been proposed as an integrative measure to quantify health status. Objective: to investigate changes in the near-term lethality of frailty before and after a 20-year interval using the frailty index (FI), a summary of age-related health deficit accumulation. Design: baseline data from the Cognitive Function and Ageing Studies (CFAS) in 1991 (n = 7,635) and 2011 (n = 7,762). Setting: three geographically distinct UK centres (Newcastle, Cambridgeshire and Nottingham). Subjects: individuals aged 65 and over (both institutionalised and community-living). Methods: a 30-item frailty score was used, which includes morbidities, risk factors and subjective measures of disability. Missing items were imputed using multiple imputations by chained equations. Binomial regression was used to investigate the relationship between frailty, age, sex and cohort. Two-year mortality was modelled using logistic regression. Results: mean frailty was slightly higher in CFAS II (0.19, 95% confidence interval (CI): 0.19-0.20) than CFAS I (0.18, 95% CI: 0.17-0.18). Two-year mortality in CFAS I was higher than in CFAS II (odds ratio (OR) = 1.16, 95% CI: 1.03-1.30). The association between frailty and 2-year mortality was non-linear with an OR of ~1.6 for each 0.10 increment in the FI. Conclusions: the relationship between frailty and mortality did not significantly differ across the studies. Severe frailty as an indicator of mortality is shown to be a stable construct.

Madagascar's grasses and grasslands: anthropogenic or natural?

Grasses, by their high productivity even under very low pCO2, their ability to survive repeated burning and to tolerate long dry seasons, have transformed the terrestrial biomes in the Neogene and Quaternary. The expansion of grasslands at the cost of biodiverse forest biomes in Madagascar is often postulated as a consequence of the Holocene settlement of the island by humans. However, we show that the Malagasy grass flora has many indications of being ancient with a long local evolutionary history, much predating the Holocene arrival of humans. First, the level of endemism in the Madagascar grass flora is well above the global average for large islands. Second, a survey of many of the more diverse areas indicates that there is a very high spatial and ecological turnover in the grass flora, indicating a high degree of niche specialization. We also find some evidence that there are both recently disturbed and natural stable grasslands: phylogenetic community assembly indicates that recently severely disturbed grasslands are phylogenetically clustered, whereas more undisturbed grasslands tend to be phylogenetically more evenly distributed. From this evidence, it is likely that grass communities existed in Madagascar long before human arrival and so were determined by climate, natural grazing and other natural factors. Humans introduced zebu cattle farming and increased fire frequency, and may have triggered an expansion of the grasslands. Grasses probably played the same role in the modification of the Malagasy environments as elsewhere in the tropics.

Age-related normative changes in phasic orthostatic blood pressure in a large population study: findings from The Irish Longitudinal Study on Ageing (TILDA).

BACKGROUND: In this report, we provide the first normative reference data and prevalence estimates of impaired orthostatic blood pressure (BP) stabilization, initial orthostatic hypotension, and orthostatic hypotension based on beat-to-beat blood pressure methods in a population-representative sample. METHODS AND RESULTS: Participants were recruited from a nationally representative cohort study (≥50 years). Beat-to-beat systolic BP, diastolic BP, and heart rate records were analyzed among those who underwent an active stand test (n=4475). Normograms were estimated by use of generalized additive models for location, shape, and scale with Box-Cox power exponential distribution. Prevalence estimates of impaired BP stabilization, initial orthostatic hypotension, and orthostatic hypotension are reported. Orthostatic BP responses in adults aged 50 to 59 years stabilized within 30 seconds of standing, with older groups taking 30 seconds or longer. The total prevalence of impaired BP stabilization was 15.6% (95% confidence interval [CI], 14.1%-17.1%), increasing with age to 41.2% (95% CI, 30.0%-52.4%) in people ≥80 years old. Initial orthostatic hypotension occurred in 32.9% (95% CI, 31.2%-34.6%) of the population aged ≥50 years, with no age gradient evident. The prevalence of orthostatic hypotension was 6.9% (95% CI, 5.9%-7.8%) in the total population, increasing to 18.5% (95% CI, 9.0%-28.0%) in those aged ≥80 years old. CONCLUSIONS: Significant age-related differences exist in the time course of postural BP responses, with abnormal responses taking longer than 30 seconds to stabilize. Impaired BP stabilization is more common as we age, affecting more than two-fifths of the population aged ≥80 years, and may play a future role in the management of falls and syncope.

Who has undiagnosed dementia? A cross-sectional analysis of participants of the Aging, Demographics and Memory Study.

BACKGROUND: delays in diagnosing dementia may lead to suboptimal care, yet around half of those with dementia are undiagnosed. Any strategy for case finding should be informed by understanding the characteristics of the undiagnosed population. We used cross-sectional data from a population-based sample with dementia aged 71 years and older in the United States to describe the undiagnosed population and identify factors associated with non-diagnosis. METHODS: the Aging, Demographics and Memory Study (ADAMS) Wave A participants (N = 856) each underwent a detailed neuropsychiatric investigation. Informants were asked whether the participant had ever received a doctor's diagnosis of dementia. We used multiple logistic regression to identify factors associated with informant report of a prior dementia diagnosis among those with a study diagnosis of dementia. RESULTS: of those with a study diagnosis of dementia (n = 307), a prior diagnosis of dementia was reported by 121 informants (weighted proportion = 42%). Prior diagnosis was associated with greater clinical dementia rating (CDR), from 26% (CDR = 1) to 83% (CDR = 5). In multivariate analysis, those aged 90 years or older were less likely to be diagnosed (P = 0.008), but prior diagnosis was more common among married women (P = 0.038) and those who had spent more than 9 years in full-time education (P = 0.043). CONCLUSIONS: people with dementia who are undiagnosed are older, have fewer years in education, are more likely to be unmarried, male and have less severe dementia than those with a diagnosis. Policymakers and clinicians should be mindful of the variation in diagnosis rates among subgroups of the population with dementia.

Using timed up and go and usual gait speed to predict incident disability in daily activities among community-dwelling adults aged 65 and older.

OBJECTIVES: To compare the ability of Timed Up and Go (TUG) and usual gait speed (UGS) to predict incident disability completing basic activities of daily living (ADL) and instrumental ADL (IADL) in older adults free of disability at baseline, and to provide estimates for the probability of incident disability at different levels of baseline mobility performance. DESIGN: Data from the first 2 waves of The Irish Longitudinal Study on Ageing, a study assessing health, economic, and social aspects of ageing in adults aged ≥50 years. SETTING: A nationally representative, population-based sample of community-dwelling adults. PARTICIPANTS: Participants aged ≥65 years who completed mobility tests during a health assessment, had no reported difficulty in ADL/IADL, and had a Mini-Mental State Examination score ≥24 were re-interviewed after 2 years (n=1664). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Participants completed the TUG and UGS at baseline and indicated difficulty in a number of basic ADL and IADL at follow-up. RESULTS: Receiver operating characteristic analysis indicated that TUG and UGS are acceptable tools to predict disability in ADL and IADL (area under the curve [AUC]=.65-.75) with no significant difference between them (P>.05). Both were excellent predictors of difficulty in higher-level functioning tasks such as preparing hot meals, taking medications, and managing money (AUC>.80). Predictive probabilities were obtained across a range of performance levels. CONCLUSIONS: TUG and UGS have similar predictive ability in relation to incident disability in basic ADL and IADL. Predictive probabilities can be used to identify those most at risk and in need of particular services. Since improving physical function can prevent or delay dependence in ADL/IADL, TUG and UGS can also provide performance goals and feedback during exercise interventions.