Ageing is generally viewed as a detrimental phenotype; with age comes increasing susceptibility to disease and frailty. Recent data also suggests that disease can result in an increase in ageing ...phenotypes suggesting a positive feedback loop. It is clear that lifespan can be modified genetically and by interventions in certain organisms but the mechanisms by which this is achieved have not yet been fully elucidated, as indeed is the case for the ageing process itself. Because of the intimate relationship between disease, ageing and ultimately lifespan it is difficult to dissect the effects of individual changes. As we learn more about individual pathways and allelic variants influencing ageing and disease we can begin to unravel the influence of natural selection on these processes.
Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative ...mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.
Online dietary assessment tools can reduce administrative costs and facilitate repeated dietary assessment during follow-up in large-scale studies. However, information on bias due to measurement ...error of such tools is limited. We developed an online 24-h recall (myfood24) and compared its performance with a traditional interviewer-administered multiple-pass 24-h recall, assessing both against biomarkers.
Metabolically stable adults were recruited and completed the new online dietary recall, an interviewer-based multiple pass recall and a suite of reference measures. Longer-term dietary intake was estimated from up to 3 × 24-h recalls taken 2 weeks apart. Estimated intakes of protein, potassium and sodium were compared with urinary biomarker concentrations. Estimated total sugar intake was compared with a predictive biomarker and estimated energy intake compared with energy expenditure measured by accelerometry and calorimetry. Nutrient intakes were also compared to those derived from an interviewer-administered multiple-pass 24-h recall.
Biomarker samples were received from 212 participants on at least one occasion. Both self-reported dietary assessment tools led to attenuation compared to biomarkers. The online tools resulted in attenuation factors of around 0.2-0.3 and partial correlation coefficients, reflecting ranking intakes, of approximately 0.3-0.4. This was broadly similar to the more administratively burdensome interviewer-based tool. Other nutrient estimates derived from myfood24 were around 10-20% lower than those from the interviewer-based tool, with wide limits of agreement. Intraclass correlation coefficients were approximately 0.4-0.5, indicating consistent moderate agreement.
Our findings show that, whilst results from both measures of self-reported diet are attenuated compared to biomarker measures, the myfood24 online 24-h recall is comparable to the more time-consuming and costly interviewer-based 24-h recall across a range of measures.
Coherent components in the dynamics of decay of stimulated emission from the primary electron donor excited state P*, and of population of the product charge-separated states
P
+
B
A
-
and
P
+
H
A
-
..., were studied in GM203L mutant reaction centers (RCs) of
Rhodobacter (Rb.) sphaeroides by measuring oscillations in the kinetics of absorbance changes at 940
nm (P* stimulated emission region), 1020
nm (
B
A
-
absorption region) and 760
nm (H
A bleaching region). Absorbance changes were induced by excitation of P (870
nm) with 18
fs pulses at 90
K. In the GM203L mutant, replacement of Gly M203 by Leu results in exclusion of the crystallographically defined water molecule (HOH55) located close to the oxygen of the 13
1-keto carbonyl group of B
A and to His M202, which provides the axial ligand to the Mg of the P
B bacteriochlorophyll. The results of femtosecond measurements were compared with those obtained with
Rb. sphaeroides R-26 RCs containing an intact water HOH55. The main consequences of the GM203L mutation were found to be as follows: (i) a low-frequency oscillation at 32
cm
−1, which is characteristic of the HOH55-containing RCs, disappears from the kinetics of absorbance changes at 1020 and 760
nm in the mutant RC; (ii) electron transfer from P* to B
A in the wild type RC was characterized by two time constants of 1.1
ps (80%) and 4.3
ps (20%), but in the GM203L mutant was characterized by a single time constant of 4.3
ps, demonstrating a slowing of primary charge separation. The previously postulated rotation of water HOH55 with a fundamental frequency of 32
cm
−1, triggered by electron transfer from P* to B
A, was confirmed by observation of an isotopic shift of the 32
cm
−1 oscillation in the kinetics of
P
+
B
A
-
population in deuterated, pheophytin-modified RCs of
Rb. sphaeroides R-26, by a factor of 1.6. These data are discussed in terms of the influence of water HOH55 on the energetics of the
P
∗
→
P
+
B
A
-
reaction, and protein dynamic events that occur on the time scale of this reaction.
HLA‐DM is an MHC class II‐related heterodimer that is targeted to lysosomal compartments by a tyrosine‐based signal YTPL, present in the cytoplasmic tail of the β chain. Similar signals in other ...proteins control transport to different intracellular locations and can be recognized at several sorting sites within the cell including the trans‐Golgi network, the plasma membrane and the early or sorting endosome. Therefore, in addition to recognizing the basic tyrosine motif, the sorting machinery must be sensitive to additional features associated with these elements. Here we show that efficient trafficking of HLA‐DM to lysosomal compartments is dependent upon the proximity of its tyrosine motif to the transmembrane domain. Constructs in which the spacing is altered are rapidly internalized but are expressed at the cell surface. We conclude that the spacing of the HLA‐DMB‐encoded tyrosine motif relative to the transmembrane domain is an important feature controlling DM sorting in endosomes.
The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and ...affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide
. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy
. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite
. Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance
, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively
, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.
Which Frail Older People Are Dehydrated? The UK DRIE Study Hooper, Lee; Bunn, Diane K; Downing, Alice ...
The journals of gerontology. Series A, Biological sciences and medical sciences,
10/2016, Letnik:
71, Številka:
10
Journal Article
Recenzirano
Odprti dostop
Water-loss dehydration in older people is associated with increased mortality and disability. We aimed to assess the prevalence of dehydration in older people living in UK long-term care and ...associated cognitive, functional, and health characteristics.
The Dehydration Recognition In our Elders (DRIE) cohort study included people aged 65 or older living in long-term care without heart or renal failure. In a cross-sectional baseline analysis, we assessed serum osmolality, previously suggested dehydration risk factors, general health, markers of continence, cognitive and functional health, nutrition status, and medications. Univariate linear regression was used to assess relationships between participant characteristics and serum osmolality, then associated characteristics entered into stepwise backwards multivariate linear regression.
DRIE included 188 residents (mean age 86 years, 66% women) of whom 20% were dehydrated (serum osmolality >300 mOsm/kg). Linear and logistic regression suggested that renal, cognitive, and diabetic status were consistently associated with serum osmolality and odds of dehydration, while potassium-sparing diuretics, sex, number of recent health contacts, and bladder incontinence were sometimes associated. Thirst was not associated with hydration status.
DRIE found high prevalence of dehydration in older people living in UK long-term care, reinforcing the proposed association between cognitive and renal function and hydration. Dehydration is associated with increased mortality and disability in older people, but trials to assess effects of interventions to support healthy fluid intakes in older people living in residential care are needed to enable us to formally assess causal direction and any health benefits of increasing fluid intakes.
Aims/hypothesis
Hypoglycaemia is a major barrier to good glucose control in type 1 diabetes. Frequent hypoglycaemic episodes impair awareness of subsequent hypoglycaemic bouts. Neural changes ...underpinning awareness of hypoglycaemia are poorly defined and molecular mechanisms by which glial cells contribute to hypoglycaemia sensing and glucose counterregulation require further investigation. The aim of the current study was to examine whether, and by what mechanism, human primary astrocyte (HPA) function was altered by acute and recurrent low glucose (RLG).
Methods
To test whether glia, specifically astrocytes, could detect changes in glucose, we utilised HPA and U373 astrocytoma cells and exposed them to RLG in vitro. This allowed measurement, with high specificity and sensitivity, of RLG-associated changes in cellular metabolism. We examined changes in protein phosphorylation/expression using western blotting. Metabolic function was assessed using a Seahorse extracellular flux analyser. Immunofluorescent imaging was used to examine cell morphology and enzymatic assays were used to measure lactate release, glycogen content, intracellular ATP and nucleotide ratios.
Results
AMP-activated protein kinase (AMPK) was activated over a pathophysiologically relevant glucose concentration range. RLG produced an increased dependency on fatty acid oxidation for basal mitochondrial metabolism and exhibited hallmarks of mitochondrial stress, including increased proton leak and reduced coupling efficiency. Relative to glucose availability, lactate release increased during low glucose but this was not modified by RLG. Basal glucose uptake was not modified by RLG and glycogen levels were similar in control and RLG-treated cells. Mitochondrial adaptations to RLG were partially recovered by maintaining euglycaemic levels of glucose following RLG exposure.
Conclusions/interpretation
Taken together, these data indicate that HPA mitochondria are altered following RLG, with a metabolic switch towards increased fatty acid oxidation, suggesting glial adaptations to RLG involve altered mitochondrial metabolism that could contribute to defective glucose counterregulation to hypoglycaemia in diabetes.
InterPro (http://www.ebi.ac.uk/interpro/) is a freely available database used to classify protein sequences into families and to predict the presence of important domains and sites. InterProScan is ...the underlying software that allows both protein and nucleic acid sequences to be searched against InterPro's predictive models, which are provided by its member databases. Here, we report recent developments with InterPro and its associated software, including the addition of two new databases (SFLD and CDD), and the functionality to include residue-level annotation and prediction of intrinsic disorder. These developments enrich the annotations provided by InterPro, increase the overall number of residues annotated and allow more specific functional inferences.
Abstract
The InterPro database (http://www.ebi.ac.uk/interpro/) classifies protein sequences into families and predicts the presence of functionally important domains and sites. Here, we report ...recent developments with InterPro (version 70.0) and its associated software, including an 18% growth in the size of the database in terms on new InterPro entries, updates to content, the inclusion of an additional entry type, refined modelling of discontinuous domains, and the development of a new programmatic interface and website. These developments extend and enrich the information provided by InterPro, and provide greater flexibility in terms of data access. We also show that InterPro's sequence coverage has kept pace with the growth of UniProtKB, and discuss how our evaluation of residue coverage may help guide future curation activities.
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