Development and validation of predictive risk models for sight threatening diabetic retinopathy in patients with type 2 diabetes to be applied as triage tools in resource limited settings.

Background: Delayed diagnosis and treatment of sight threatening diabetic retinopathy (STDR) is a common cause of visual impairment in people with Type 2 diabetes. Therefore, systematic regular retinal screening is recommended, but global coverage of such services is challenging. We aimed to develop and validate predictive models for STDR to identify 'at-risk' population for retinal screening. Methods: Models were developed using datasets obtained from general practices in inner London, United Kingdom (UK) on adults with type 2 Diabetes during the period 2007-2017. Three models were developed using Cox regression and model performance was assessed using C statistic, calibration slope and observed to expected ratio measures. Models were externally validated in cohorts from Wales, UK and India. Findings: A total of 40,334 people were included in the model development phase of which 1427 (3·54%) people developed STDR. Age, gender, diabetes duration, antidiabetic medication history, glycated haemoglobin (HbA1c), and history of retinopathy were included as predictors in the Model 1, Model 2 excluded retinopathy status, and Model 3 further excluded HbA1c. All three models attained strong discrimination performance in the model development dataset with C statistics ranging from 0·778 to 0·832, and in the external validation datasets (C statistic 0·685 - 0·823) with calibration slopes closer to 1 following re-calibration of the baseline survival. Interpretation: We have developed new risk prediction equations to identify those at risk of STDR in people with type 2 diabetes in any resource-setting so that they can be screened and treated early. Future testing, and piloting is required before implementation. Funding: This study was funded by the GCRF UKRI (MR/P207881/1) and supported by the NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology.

Development and validation of resource-driven risk prediction models for incident chronic kidney disease in type 2 diabetes.

Prediction models for population-based screening need, for global usage, to be resource-driven, involving predictors that are affordably resourced. Here, we report the development and validation of three resource-driven risk models to identify people with type 2 diabetes (T2DM) at risk of stage 3 CKD defined by a decline in estimated glomerular filtration rate (eGFR) to below 60 mL/min/1.73m2. The observational study cohort used for model development consisted of data from a primary care dataset of 20,510 multi-ethnic individuals with T2DM from London, UK (2007-2018). Discrimination and calibration of the resulting prediction models developed using cox regression were assessed using the c-statistic and calibration slope, respectively. Models were internally validated using tenfold cross-validation and externally validated on 13,346 primary care individuals from Wales, UK. The simplest model was simplified into a risk score to enable implementation in community-based medicine. The derived full model included demographic, laboratory parameters, medication-use, cardiovascular disease history (CVD) and sight threatening retinopathy status (STDR). Two less resource-intense models were developed by excluding CVD and STDR in the second model and HbA1c and HDL in the third model. All three 5-year risk models had good internal discrimination and calibration (optimism adjusted C-statistics were each 0.85 and calibration slopes 0.999-1.002). In Wales, models achieved excellent discrimination(c-statistics ranged 0.82-0.83). Calibration slopes at 5-years suggested models over-predicted risks, however were successfully updated to accommodate reduced incidence of stage 3 CKD in Wales, which improved their alignment with the observed rates in Wales (E/O ratios near to 1). The risk score demonstrated similar model performance compared to direct evaluation of the cox model. These resource-driven risk prediction models may enable universal screening for Stage 3 CKD to enable targeted early optimisation of risk factors for CKD.

Impact of dietary protein on postprandial glycaemic control and insulin requirements in Type 1 diabetes: a systematic review.

AIM: Postprandial hyperglycaemia is a challenge for people living with Type 1 diabetes. In addition to carbohydrate, dietary protein has been shown to contribute to postprandial glycaemic excursions with recommendations to consider protein when calculating mealtime insulin doses. The aim of this review is to identify and synthesize evidence about the glycaemic impact of dietary protein and insulin requirements for individuals with Type 1 diabetes. METHODS: A systematic literature search of relevant biomedical databases was performed to identify research on the glycaemic impact of dietary protein when consumed alone, and in combination with other macronutrients in individuals with Type 1 diabetes. RESULTS: The review included 14 published studies dated from 1992 to 2018, and included studies that researched the impact of protein alone (n = 2) and protein in a mixed meal (n = 12). When protein was consumed alone a glycaemic effect was not seen until ≥ 75 g. In a carbohydrate-containing meal ≥ 12.5 g of protein impacted the postprandial glucose. Inclusion of fat in a high-protein meal enhanced the glycaemic response and further increased insulin requirements. The timing of the glycaemic effect from dietary protein ranged from 90 to 240 min. Studies indicate that the postprandial glycaemic response and insulin requirements for protein are different when protein is consumed alone or with carbohydrate and/or fat. CONCLUSIONS: This systematic review provides evidence that dietary protein contributes to postprandial glycaemic excursions and insulin requirements. These insights have important implications for the education of people with Type 1 diabetes and highlights the need for more effective insulin dosing strategies for mixed macronutrient meals.

Detection of workplace sedentary behavior using thermal sensors.

Sedentary behavior has been linked to leading causes of morbidity and mortality, including cancer, cardiovascular disease and diabetes. Those who work in office workplaces are susceptible to higher levels of sedentary behavior during the working day. This paper introduces a novel approach to the detection of sedentary behavior through the use of a thermal sensor mounted on the ceiling above a busy workspace. This solution was found to more accurately record 7 out of 10 activity metrics in comparison to self-assessment, when compared to chair pressure sensor recordings.

The effect of different doses of an arginine-containing supplement on the healing of pressure ulcers.

OBJECTIVE: To investigate if a lower dose of arginine in the form of an oral nutritional supplement can show similar benefit in the healing rate of pressure ulcers compared with the current evidence for 9g of arginine. METHOD: Twenty-three inpatients with category II, III or IV pressure ulcers were randomised to receive daily, for 3 weeks, the standard hospital diet plus 4.5 or 9g arginine in the form of a commercial supplement. Pressure ulcer size and severity was measured weekly (by PUSH tool; pressure ulcer scale for healing; 0= completely healed, 17= greatest severity). Nutritional status was determined by Subjective Global Assessment. RESULTS: There were no significant differences in patients' age, gender, BMI, haemoglobin levels, albumin levels and diagnosis of diabetes between treatment groups. There was a significant decrease in pressure ulcer severity over time (p < 0.001), with no evidence of a difference in healing rate between the two arginine dosages (p=0.991). Based on expected healing time, patients in both treatment groups were estimated to achieve an almost 2-fold improvement compared with the historical control group. Patients categorised as malnourished showed clinically significant impaired healing rates compared with well nourished patients (p=0.057), although this was unaffected by arginine dosage (p=0.727). CONCLUSION: Similar clinical benefits in healing of pressure ulcers can be achieved with a lower dosage of arginine, which can translate into improved concordance and significant cost-savings for both the health-care facilities and for patients.

Phosphorylation of the voltage-gated potassium channel Kv2.1 by AMP-activated protein kinase regulates membrane excitability.

Firing of action potentials in excitable cells accelerates ATP turnover. The voltage-gated potassium channel Kv2.1 regulates action potential frequency in central neurons, whereas the ubiquitous cellular energy sensor AMP-activated protein kinase (AMPK) is activated by ATP depletion and protects cells by switching off energy-consuming processes. We show that treatment of HEK293 cells expressing Kv2.1 with the AMPK activator A-769662 caused hyperpolarizing shifts in the current-voltage relationship for channel activation and inactivation. We identified two sites (S440 and S537) directly phosphorylated on Kv2.1 by AMPK and, using phosphospecific antibodies and quantitative mass spectrometry, show that phosphorylation of both sites increased in A-769662-treated cells. Effects of A-769662 were abolished in cells expressing Kv2.1 with S440A but not with S537A substitutions, suggesting that phosphorylation of S440 was responsible for these effects. Identical shifts in voltage gating were observed after introducing into cells, via the patch pipette, recombinant AMPK rendered active but phosphatase-resistant by thiophosphorylation. Ionomycin caused changes in Kv2.1 gating very similar to those caused by A-769662 but acted via a different mechanism involving Kv2.1 dephosphorylation. In cultured rat hippocampal neurons, A-769662 caused hyperpolarizing shifts in voltage gating similar to those in HEK293 cells, effects that were abolished by intracellular dialysis with Kv2.1 antibodies. When active thiophosphorylated AMPK was introduced into cultured neurons via the patch pipette, a progressive, time-dependent decrease in the frequency of evoked action potentials was observed. Our results suggest that activation of AMPK in neurons during conditions of metabolic stress exerts a protective role by reducing neuronal excitability and thus conserving energy.

Selective expression in carotid body type I cells of a single splice variant of the large conductance calcium- and voltage-activated potassium channel confers regulation by AMP-activated protein kinase.

Inhibition of large conductance calcium-activated potassium (BKCa) channels mediates, in part, oxygen sensing by carotid body type I cells. However, BKCa channels remain active in cells that do not serve to monitor oxygen supply. Using a novel, bacterially derived AMP-activated protein kinase (AMPK), we show that AMPK phosphorylates and inhibits BKCa channels in a splice variant-specific manner. Inclusion of the stress-regulated exon within BKCa channel α subunits increased the stoichiometry of phosphorylation by AMPK when compared with channels lacking this exon. Surprisingly, however, the increased phosphorylation conferred by the stress-regulated exon abolished BKCa channel inhibition by AMPK. Point mutation of a single serine (Ser-657) within this exon reduced channel phosphorylation and restored channel inhibition by AMPK. Significantly, RT-PCR showed that rat carotid body type I cells express only the variant of BKCa that lacks the stress-regulated exon, and intracellular dialysis of bacterially expressed AMPK markedly attenuated BKCa currents in these cells. Conditional regulation of BKCa channel splice variants by AMPK may therefore determine the response of carotid body type I cells to hypoxia.

Ion channel regulation by AMPK: the route of hypoxia-response coupling in thecarotid body and pulmonary artery.

Vital homeostatic mechanisms monitor O2 supply and adjust respiratory and circulatory function to meet demand. The pulmonary arteries and carotid bodies are key systems in this respect. Hypoxic pulmonary vasoconstriction (HPV) aids ventilation-perfusion matching in the lung by diverting blood flow from areas with an O2 deficit to those rich in O2, while a fall in arterial pO2 increases sensory afferent discharge from the carotid body to elicit corrective changes in breathing patterns. We discuss here the new concept that hypoxia, by inhibiting oxidative phosphorylation, activates AMP-activated protein kinase (AMPK) leading to consequent phosphorylation of target proteins, such as ion channels, which initiate pulmonary artery constriction and carotid body activation. Consistent with this view, AMPK knockout mice exhibit an impaired ventilatory response to hypoxia. Thus, AMPK may be sufficient and necessary for hypoxia-response coupling and may regulate O2 and thereby energy (ATP) supply at the whole body as well as the cellular level.

Crystallization of the Bacillus subtilis SPP1 bacteriophage helicase loader protein G39P.

The essential helicase loader protein G39P encoded by Bacillus subtilis SPP1 phage has been overproduced in Escherichia coli and purified. The wild-type protein has been crystallized by the hanging-drop vapour-diffusion method in a primitive hexagonal space group, probably P6(1)22/P6(5)22, but the crystals diffract to only 3.4 A and are poorly reproducible. Mass-spectrometric analysis has revealed marked proteolytic cleavage from the C-terminus and the presence of a major species corresponding to deletion of the 14 C-terminal residues. Thus, a new variant of the protein (G39P112) has been engineered that corresponds to a 14-residue C-terminal truncation. The G39P112 variant has also been crystallized but now in a primitive orthorhombic form, probably P2(1)2(1)2 or P2(1)2(1)2(1), with unit-cell parameters a = 85.6, b = 89.7, c = 47.6 A, with diffraction to 2.4 A on a synchrotron source and with greatly improved reproducibility. Calculation of V(M) values for this G39P112 variant suggests the presence of three monomers in the asymmetric unit, corresponding to a solvent content of about 47%. A selenomethionine-incorporated form of the protein has been produced and a full three-wavelength MAD data collection undertaken.

Cloning, characterisation and crystallisation of a diadenosine 5',5"'-P(1),P(4)-tetraphosphate pyrophosphohydrolase from Caenorhabditis elegans.

Asymmetrically cleaving diadenosine 5',5"'-P(1),P(4)-tetraphosphate (Ap4A) hydrolase activity has been detected in extracts of adult Caenorhabditis elegans and the corresponding cDNA amplified and expressed in Escherichia coli. As expected, sequence analysis shows the enzyme to be a member of the Nudix hydrolase family. The purified recombinant enzyme behaves as a typical animal Ap4A hydrolase. It hydrolyses Ap4A with a K(m) of 7 microM and k(cat) of 27 s(-1) producing AMP and ATP as products. It is also active towards other adenosine and diadenosine polyphosphates with four or more phosphate groups, but not diadenosine triphosphate, always generating ATP as one of the products. It is inhibited non-competitively by fluoride (K(i)=25 microM) and competitively by adenosine 5'-tetraphosphate with Ap4A as substrate (K(i)=10 nM). Crystals of diffraction quality with the morphology of rectangular plates were readily obtained and preliminary data collected. These crystals diffract to a minimum d-spacing of 2 A and belong to either space group C222 or C222(1). Phylogenetic analysis of known and putative Ap4A hydrolases of the Nudix family suggests that they fall into two groups comprising plant and Proteobacterial enzymes on the one hand and animal and archaeal enzymes on the other. Complete structural determination of the C. elegans Ap4A hydrolase will help determine the basis of this grouping.

A study of the structure-activity relationship for diazaborine inhibition of Escherichia coli enoyl-ACP reductase.

Enoyl acyl carrier protein (ACP) reductase catalyses the last reductive step of fatty acid biosynthesis, reducing the enoyl group of a growing fatty acid chain attached to ACP to its acyl product using NAD(P)H as the cofactor. This enzyme is the target for the diazaborine class of antibacterial agents, the biocide triclosan, and one of the targets for the front-line anti-tuberculosis drug isoniazid. The structures of complexes of Escherichia coli enoyl-ACP reductase (ENR) from crystals grown in the presence of NAD+ and a family of diazaborine compounds have been determined. Analysis of the structures has revealed that a mobile loop in the structure of the binary complex with NAD+ becomes ordered on binding diazaborine/NAD+ but displays a different conformation in the two subunits of the asymmetric unit. The work presented here reveals how, for one of the ordered conformations adopted by the mobile loop, the mode of diazaborine binding correlates well with the activity profiles of the diazaborine family. Additionally, diazaborine binding provides insights into the pocket on the enzyme surface occupied by the growing fatty acid chain.

Assays to predict the genotoxicity of the chromosomal mutagen etoposide -- focussing on the best assay.

The topoisomerase II inhibitor etoposide is used routinely to treat a variety of cancers in patients of all ages. As a result of its extensive use in the clinic and its association with secondary malignancies it has become a compound of great interest with regard to its genotoxic activity in vivo. This paper describes a series of assays that were employed to determine the in vivo genotoxicity of etoposide in a murine model system. The alkaline comet assay detected DNA damage in the bone marrow mononuclear compartment over the dose range of 10--100mg/kg and was associated with a large and dose dependent rise in the proportion of cells with severely damaged DNA. In contrast, the bone marrow micronucleus assay was found to be sensitive to genotoxic damage between the doses of 0.1--1mg/kg without any corresponding increases in cytotoxicity. An increase in the mutant frequency was undetectable at the Hprt locus at administered doses of 1 and 10mg/kg of etoposide, however, an increase in the mutant frequency was seen at the Aprt locus at these doses. We conclude that the BMMN assay is a good short-term predictor of the clastogenicity of etoposide at doses that do not result in cytotoxic activity, giving an indication of potential mutagenic effects. Moreover, the detection of mutants at the Aprt locus gives an indication of the potential of etoposide to cause chromosomal mutations that may lead to secondary malignancy.

Crystallization and quaternary structure analysis of an Lrp-like regulatory protein from the hyperthermophile Pyrococcus furiosus.

The LrpA transcriptional regulator from Pyrococcus furiosus, a member of the leucine-responsive regulatory protein (Lrp) family, has been crystallized by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant. The crystals belong to the tetragonal system and are in space group I4(1)22, with unit-cell parameters a = b = 104.5, c = 245.1 A. Consideration of the values of V(M) and possible packing of the molecules within the cell suggest that the asymmetric unit contains a dimer. Examination of the behaviour of the protein on gel-filtration columns and analysis of the self-rotation function suggests that the molecule is an octamer in solution at around pH 5. Determination of the structure of this protein will provide insights into the mechanisms responsible for DNA-protein recognition at high temperature and into how the regulatory properties of the Lrp family are modified by the presence or absence of effector molecules.

Analysis of the structure, substrate specificity, and mechanism of squash glycerol-3-phosphate (1)-acyltransferase.

BACKGROUND: Glycerol-3-phosphate (1)-acyltransferase(G3PAT) catalyzes the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acyl-CoAs into the sn-1 position of glycerol 3-phosphate to yield 1-acylglycerol-3-phosphate. G3PATs can either be selective, preferentially using the unsaturated fatty acid, oleate (C18:1), as the acyl donor, or nonselective, using either oleate or the saturated fatty acid, palmitate (C16:0), at comparable rates. The differential substrate specificity for saturated versus unsaturated fatty acids seen within this enzyme family has been implicated in the sensitivity of plants to chilling temperatures. RESULTS: The three-dimensional structure of recombinant G3PAT from squash chloroplast has been determined to 1.9 A resolution by X-ray crystallography using the technique of multiple isomorphous replacement and provides the first representative structure of an enzyme of this class. CONCLUSIONS: The tertiary structure of G3PAT comprises two domains, the larger of which, domain II, features an extensive cleft lined by hydrophobic residues and contains at one end a cluster of positively charged residues flanked by a H(X)(4)D motif, which is conserved amongst many glycerolipid acyltransferases. We predict that these hydrophobic and positively charged residues represent the binding sites for the fatty acyl substrate and the phosphate moiety of the glycerol 3-phosphate, respectively, and that the H(X)(4)D motif is a critical component of the enzyme's catalytic machinery.

Crystal structure of the Lrp-like transcriptional regulator from the archaeon Pyrococcus furiosus.

The LrpA protein from the hyperthermophilic archaeon Pyrococcus furiosus belongs to the Lrp/AsnC family of transcriptional regulatory proteins, of which the Escherichia coli leucine-responsive regulatory protein is the archetype. Its crystal structure has been determined at 2.9 A resolution and is the first for a member of the Lrp/AsnC family, as well as one of the first for a transcriptional regulator from a hyperthermophile. The structure consists of an N-terminal domain containing a helix-turn-helix (HtH) DNA-binding motif, and a C-terminal domain of mixed alpha/beta character reminiscent of a number of RNA- and DNA-binding domains. Pyrococcus furiosus LrpA forms a homodimer mainly through interactions between the antiparallel beta-sheets of the C-terminal domain, and further interactions lead to octamer formation. The LrpA structure suggests how the protein might bind and possibly distort its DNA substrate through use of its HtH motifs and control gene expression. A possible location for an effector binding site is proposed by using sequence comparisons with other members of the family coupled to mutational analysis.

Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I.

Fab I, enoyl acyl carrier protein reductase (ENR), is an enzyme used in fatty acid synthesis. It is a single chain polypeptide in plants, bacteria, and mycobacteria, but is part of a complex polypeptide in animals and fungi. Certain other enzymes in fatty acid synthesis in apicomplexan parasites appear to have multiple forms, homologous to either a plastid, plant-like single chain enzyme or more like the animal complex polypeptide chain. We identified a plant-like Fab I in Plasmodium falciparum and modelled the structure on the Brassica napus and Escherichia coli structures, alone and complexed to triclosan (5-chloro-2-[2,4 dichlorophenoxy] phenol]), which confirmed all the requisite features of an ENR and its interactions with triclosan. Like the remarkable effect of triclosan on a wide variety of bacteria, this compound markedly inhibits growth and survival of the apicomplexan parasites P. falciparum and Toxoplasma gondii at low (i.e. IC50 congruent with150-2000 and 62 ng/ml, respectively) concentrations. Discovery and characterisation of an apicomplexan Fab I and discovery of triclosan as lead compound provide means to rationally design novel inhibitory compounds.

The effects of dose, route of administration, drug scheduling and MDR-1 gene transfer on the genotoxicity of etoposide in bone marrow.

We have used the bone marrow micronucleus assay (BMMN) as a measure of clastogenicity, in response to etoposide exposure in murine bone marrow. Oral delivery of etoposide resulted in a reduced number of micronucleated polychromatic erythrocytes (MPE) relative to the same dose delivered intraperitoneally (P < 0.001). Daily fractionation of the oral schedule of etoposide led to a more than six-fold increase in cumulative MPE frequency over that observed with the same total, unfractionated dose, with the potency of the response increasing with serial exposure (r = 0.79). Retrovirally-mediated expression of MDR1 in murine bone marrow resulted in partial protection against the clastogenic activity of etoposide relative to mock transduced control mice. The model system developed has indicated a variety of factors able to influence the genotoxicity of etoposide. It should now be possible to further exploit this model in order to define other factors governing haemopoietic sensitivity to etoposide.