Effects of molecular crowding on structural and dynamical properties of biological macromolecules do depend on the concentration of crowding agents but also on the molecular mass and the structural ...compactness of the crowder molecules. By employing fluorescence correlation spectroscopy (FCS), we investigated the translational mobility of several biological macromolecules ranging from 17 kDa to 2.7 MDa. Polyethylene glycol and Ficoll polymers of different molecular masses were used in buffer solutions to mimic a crowded environment. The reduction in translational mobility of the biological tracer molecules was analyzed as a function of crowder volume fractions and was generally more pronounced in PEG as compared to Ficoll solutions. For several crowding conditions, we observed a molecular sieving effect, in which the diffusion coefficient of larger tracer molecules is reduced to a larger extent than predicted by the Stokes–Einstein relation. By employing a FRET-based biosensor, we also showed that a multiprotein complex is significantly compacted in the presence of macromolecular crowders. Importantly, with respect to sensor in vivo applications, ligand concentration determining sensors would need a crowding specific calibration in order to deliver correct cytosolic ligand concentration.
Inspired by the modular architecture of natural signaling proteins, ligand binding proteins are equipped with two fluorescent proteins (FPs) in order to obtain Förster resonance energy transfer ...(FRET)-based biosensors. Here, we investigated a glucose sensor where the donor and acceptor FPs were attached to a glucose binding protein using a variety of different linker sequences. For three resulting sensor constructs the corresponding glucose induced conformational changes were measured by small angle X-ray scattering (SAXS) and compared to recently published single molecule FRET results (Höfig et al., ACS Sensors, 2018). For one construct which exhibits a high change in energy transfer and a large change of the radius of gyration upon ligand binding, we performed coarse-grained molecular dynamics simulations for the ligand-free and the ligand-bound state. Our analysis indicates that a carefully designed attachment of the donor FP is crucial for the proper transfer of the glucose induced conformational change of the glucose binding protein into a well pronounced FRET signal change as measured in this sensor construct. Since the other FP (acceptor) does not experience such a glucose induced alteration, it becomes apparent that only one of the FPs needs to have a well-adjusted attachment to the glucose binding protein.
This study is a secondary analysis of a randomized controlled trial using Paleolithic diet and exercise in individuals with type 2 diabetes. We hypothesized that increased adherence to the ...Paleolithic diet was associated with greater effects on blood pressure, blood lipids and HbA1c independent of weight loss. Participants were asked to follow a Paleolithic diet for 12 weeks and were randomized to supervised exercise or general exercise recommendations. Four-day food records were analyzed, and food items characterized as "Paleolithic" or "not Paleolithic". Foods considered Paleolithic were lean meat, poultry, fish, seafood, fruits, nuts, berries, seeds, vegetables, and water to drink; "not Paleolithic" were legumes, cereals, sugar, salt, processed foods, and dairy products. A Paleo ratio was calculated by dividing the Paleolithic calorie intake by total calorie intake. A multiple regression model predicted the outcome at 12 weeks using the Paleo ratio, group affiliation, and outcome at baseline as predictors. The Paleo ratio increased from 28% at baseline to 94% after the intervention. A higher Paleo ratio was associated with lower fat mass, BMI, waist circumference, systolic blood pressure, and serum triglycerides at 12 weeks, but not with lower HbA1c levels. The Paleo ratio predicted triglyceride levels independent of weight loss (
= 0.046). Moreover, an increased monounsaturated/saturated fatty acids ratio and an increased polyunsaturated/saturated fatty acids ratio was associated with lower triglyceride levels independent of weight loss. (
= 0.017 and
= 0.019 respectively). We conclude that a higher degree of adherence to the Paleolithic diet recommendations improved fat quality and was associated with improved triglyceride levels independent of weight loss among individuals with type 2 diabetes.
ObjectiveTo assess the effect of adding an oral glucose tolerance test (OGTT) to fasting plasma glucose (FPG) in terms of detection of type 2 diabetes (T2D) and impaired glucose tolerance ...(IGT).DesignRetrospective analysis of serial cross-sectional screening study.SettingPopulation-based health examinations within primary care in Västerbotten County, Sweden.ParticipantsIndividuals aged 40- 50 and 60 years with participation from 1985 to 2017. Those with previously diagnosed diabetes and FPG≥7 mmol/L were excluded.Primary and secondary outcome measuresPrevalence of hyperglycaemia on the OGTT (IGT and T2D defined as 2-hour postload capillary plasma glucose of 8.9–12.1 mmol/L and ≥12.2 mmol/L, respectively). Analyses were further stratified by age, sex and risk factor burden to identify groups at high or low risk of IGT and T2D on testing. The numbers needed to screen (NNS) to prevent one case of T2D through detection and treatment of IGT was estimated, combining prevalence numbers with average progression rates and intervention effects from previous meta-analyses.ResultsThe prevalence of IGT ranged from 0.9% (95% CI 0.7% to 1.1%) to 29.6% (95% CI 27.4% to 31.7%), and the prevalence of T2D ranged from 0.06% (95% CI 0.02% to 0.11%) to 7.0% (95% CI 5.9% to 8.3%), depending strongly on age, sex and risk factor burden. The estimated NNS to prevent one case of T2D through detection and lifestyle treatment of IGT ranged from 1332 among 40-year-old men without risk factors, to 39 among 60-year-old women with all risk factors combined.ConclusionsThe prevalence of hyperglycaemia on OGTT is highly dependent on age, sex and risk factor burden; OGTT should be applied selectively to high-risk groups to avoid unnecessary testing in the general population.
To describe the process of becoming aware of and acting on personal cardiovascular (CVD) risk in type 2 diabetes (T2D).
A purposive sample of 14 persons living with T2D participated in ...semi-structured, open-ended, in-dept interviews. The interviews were analysed with grounded theory.
The analysis identified the core category "Balancing emotions, integrating knowledge and understanding to achieve risk awareness and act on it." Five categories describe the movement from not being aware of the risk of cardiovascular disease (CVD) to becoming aware of this risk and taking action to reduce it. Persons with T2D need to transform their knowledge and experience of CVD risk and incorporate it in their individual situations. Emotional and existential experiences of CVD risk can lead to awareness about the severity of the condition and contribute to increased motivation for self-management. However, an overly high emotional response can be overwhelming and may result in insufficient self-management.
Persons with T2D seemed not to fully grasp their increased risk of CVD or recognize that self-management activities were aimed at reducing this risk. However, their awareness of CVD risk gradually increased as they came to understand the severity of T2D and became more emotionally and existentially engaged.
In most microbial cultivations D-glucose is the main carbon and energy source. However, quantification of D-glucose especially in small scale is still challenging. Therefore, we developed a ...FRET-based glucose biosensor, which can be applied in microbioreactor-based cultivations. This sensor consists of a glucose binding protein sandwiched between two fluorescent proteins, constituting a FRET pair. Upon D-glucose binding the sensor undergoes a conformational change which is translated into a FRET-ratio change.
The selected sensor shows an apparent K
below 1.5 mM D-glucose and a very high sensitivity of up to 70% FRET-ratio change between the unbound and the glucose-saturated state. The soluble sensor was successfully applied online to monitor the glucose concentration in an Escherichia coli culture. Additionally, this sensor was utilized in an at-line process for a Corynebacterium glutamicum culture as an example for a process with cell-specific background (e.g. autofluorescence) and medium-induced quenching. Immobilization of the sensor via HaloTag
enabled purification and covalent immobilization in one step and increased the stability during application, significantly.
A FRET-based glucose sensor was used to quantify D-glucose consumption in microtiter plate based cultivations. To the best of our knowledge, this is the first method reported for online quantification of D-glucose in microtiter plate based cultivations. In comparison to D-glucose analysis via an enzymatic assay and HPLC, the sensor performed equally well, but enabled much faster measurements, which allowed to speed up microbial strain development significantly.
Type 2 diabetes is associated with impaired episodic memory functions and increased risk of different dementing disorders. Diet and exercise may potentially reverse these impairments. In this study, ...sedentary individuals with type 2 diabetes treated by lifestyle ± metformin were randomized to a Paleolithic diet (PD,
= 12) with and without high intensity exercise (PDEX,
= 12) for 12 weeks. Episodic memory function, associated functional brain responses and hippocampal gray matter volume was measured by magnetic resonance imaging. A matched, but not randomized, non-interventional group was included as a reference (
= 6). The PD included a high intake of unsaturated fatty acids and protein, and excluded the intake of dairy products, grains, refined sugar and salt. The exercise intervention consisted of 180 min of supervised aerobic and resistance exercise per week. Both interventions induced a significant weight loss, improved insulin sensitivity and increased peak oxygen uptake without any significant group differences. Furthermore, both interventions were associated with increased functional brain responses within the right anterior hippocampus, right inferior occipital gyrus and increased volume of the right posterior hippocampus. There were no changes in memory performance. We conclude that life-style modification may improve neuronal plasticity in brain areas linked to cognitive function in type 2 diabetes. Putative long-term effects on cognitive functions including decreased risk of dementing disorders await further studies. Clinical trials registration number: Clinicaltrials. gov NCT01513798.
Abstract
Background
Patients with type 2 diabetes have an increased risk of death and cardiovascular events and people with diabetes or prediabetes have been found to have increased atherosclerotic ...burden in the coronary and carotid arteries. This study will estimate the cross-sectional prevalence of atherosclerosis in the coronary and carotid arteries in individuals with prediabetes and diabetes, compared with normoglycaemic individuals in a large population-based cohort.
Methods
The 30,154 study participants, 50–64 years, were categorized according to their fasting glycaemic status or self-reported data as normoglycaemic, prediabetes, and previously undetected or known diabetes. Prevalence of affected coronary artery segments, severity of stenosis and coronary artery calcium score (CACS) were determined by coronary computed tomography angiography. Total atherosclerotic burden was assessed in the 11 clinically most relevant segments using the Segment Involvement Score and as the presence of any coronary atherosclerosis. The presence of atherosclerotic plaque in the carotid arteries was determined by ultrasound examination.
Results
Study participants with prediabetes (n = 4804, 16.0%) or diabetes (n = 2282, 7.6%) had greater coronary artery plaque burden, more coronary stenosis and higher CACS than normoglycaemic participants (all, p < 0.01). Among male participants with diabetes 35.3% had CACS ≥ 100 compared to 16.1% among normoglycaemic participants. For women, the corresponding figures were 8.9% vs 6.1%. The prevalence of atherosclerosis in the coronary arteries was higher in participants with previously undetected diabetes than prediabetes, but lower than in patients with known diabetes. The prevalence of any plaque in the carotid arteries was higher in participants with prediabetes or diabetes than in normoglycaemic participants.
Conclusions
In this large population-based cohort of currently asymptomatic people, the atherosclerotic burden in the coronary and carotid arteries increased with increasing degree of dysglycaemia. The finding that the atherosclerotic burden in the coronary arteries in the undetected diabetes category was midway between the prediabetes category and patients with known diabetes may have implications for screening strategies and tailored prevention interventions for people with dysglycaemia in the future.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized ...systems create a need for new rapid online detection systems to quantify the respective target compound. In this regard, biosensors, especially genetically encoded Förster resonance energy transfer (FRET)-based biosensors, offer tremendous opportunities. As a proof-of-concept, we have created a toolbox of FRET-based biosensors for the ratiometric determination of l-lysine in fermentation broth.
: The sensor toolbox was constructed based on a sensor that consists of an optimized central lysine-/arginine-/ornithine-binding protein (LAO-BP) flanked by two fluorescent proteins (enhanced cyan fluorescent protein (ECFP), Citrine). Further sensor variants with altered affinity and sensitivity were obtained by circular permutation of the binding protein as well as the introduction of flexible and rigid linkers between the fluorescent proteins and the LAO-BP, respectively.
The sensor prototype was applied to monitor the extracellular l-lysine concentration of the l-lysine producing
(
) strain DM1933 in a BioLector
microscale cultivation device. The results matched well with data obtained by HPLC analysis and the Ninhydrin assay, demonstrating the high potential of FRET-based biosensors for high-throughput microbial bioprocess optimization.
Lifestyle interventions with weight loss can improve insulin sensitivity in type 2 diabetes (T2D), but mechanisms are unclear. We explored circulating and skeletal muscle metabolite signatures of ...altered peripheral (pIS) and hepatic insulin sensitivity (hIS) in overweight and obese T2D individuals that were randomly assigned a 12-week Paleolithic-type diet with (diet-ex,
= 13) or without (diet,
= 13) supervised exercise. Baseline and post-intervention measures included: mass spectrometry-based metabolomics and lipidomics of skeletal muscle and plasma; pIS and hIS; ectopic lipid deposits in the liver and skeletal muscle; and skeletal muscle fat oxidation rate. Both groups lowered BMI and total % fat mass and increased their pIS. Only the diet-group improved hIS and reduced ectopic lipids in the liver and muscle. The combined improvement in pIS and hIS in the diet-group were associated with decreases in muscle and circulating branched-chain amino acid (BCAA) metabolites, specifically valine. Improved pIS with diet-ex was instead linked to increased diacylglycerol (34:2) and triacylglycerol (56:0) and decreased phosphatidylcholine (34:3) in muscle coupled with improved muscle fat oxidation rate. This suggests a tissue crosstalk involving BCAA-metabolites after diet intervention with improved pIS and hIS, reflecting reduced lipid influx. Increased skeletal muscle lipid utilization with exercise may prevent specific lipid accumulation at sites that perturb insulin signaling.
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