Very Early Loading of a Bone-Anchored Hearing System Is Safe.

OBJECTIVE: To investigate the feasibility of early sound processor loading with two separate bone-anchored hearing aid implants 1 to 2 weeks after implantation. Secondary endpoints included registration of any postoperative complications, skin reactions, and any adverse events. STUDY DESIGN: Prospective, nonrandomized, noncontrolled, multicenter clinical trial. SETTING: Tertiary referral center. PATIENTS: Sixty adult patients, eligible for bone-anchored hearing aid surgery, were enrolled. INTERVENTION: Implantation of the bone-anchored hearing aid implants were done using minimally invasive surgery with tissue preservation. MAIN OUTCOME MEASURE: Implant stability quotient (ISQ) values were recorded using resonance frequency analysis. Follow-ups were scheduled 5 to 12 days, 4 to 8 weeks, 6 months, and 12 months postoperatively. Differences between the lowest ISQ values (ISQ low) over time were analyzed by paired-samples t test with a significance level of 0.05. Skin and soft tissue reactions were assessed according to Holger's classification. RESULTS: Loading time of the Ponto Wide implant was 11.6 ± 1.4 days (SD) in average (median, 12 d; range, 7-19 d). Loading of the Ponto BHX implant was done 9.7 ± 3.5 days (SD) postoperatively (median, 9 d; range, 5-19 d). The implant stability increased significantly during the 12-month study period with both implants. One Ponto BHX implant was lost. Skin and soft tissue reactions were few in both groups. Across all planned follow-up visits, no skin and soft tissue reactions were observed in 86% (Ponto BHX implant) and 85% (Ponto Wide implant). CONCLUSION: Very early loading of sound processors after percutaneous bone-anchored hearing system surgery is safe already 1 to 2 weeks postoperatively.

Circulating metabolomic markers in association with overall burden of microvascular complications in type 1 diabetes.

INTRODUCTION: Diabetic retinopathy (DR), diabetic kidney disease (DKD) and distal symmetric polyneuropathy (DSPN) share common pathophysiology and pose an additive risk of early mortality. RESEARCH DESIGN AND METHODS: In adults with type 1 diabetes, 49 metabolites previously associated with either DR or DKD were assessed in relation to presence of DSPN. Metabolites overlapping in significance with presence of all three complications were assessed in relation to microvascular burden severity (additive number of complications-ie, presence of DKD±DR±DSPN) using linear regression models. Subsequently, the same metabolites were assessed with progression to endpoints: soft microvascular events (progression in albuminuria grade, ≥30% estimated glomerular filtration rate (eGFR) decline, or any progression in DR grade), hard microvascular events (progression to proliferative DR, chronic kidney failure, or ≥40% eGFR decline), and hard microvascular or macrovascular events (hard microvascular events, cardiovascular events (myocardial infarction, stroke, or arterial interventions), or cardiovascular mortality), using Cox models. All models were adjusted for sex, baseline age, diabetes duration, systolic blood pressure, HbA1c, body mass index, total cholesterol, smoking, and statin treatment. RESULTS: The full cohort investigated consisted of 487 participants. Mean (SD) follow-up was 4.8 (2.9, 5.7) years. Baseline biothesiometry was available in 202 participants, comprising the cross-sectional cohort. Eight metabolites were significantly associated with presence of DR, DKD, and DSPN, and six with additive microvascular burden severity. In the full cohort longitudinal analysis, higher levels of 3,4-dihydroxybutanoic acid (DHBA), 2,4-DHBA, ribonic acid, glycine, and ribitol were associated with development of events in both crude and adjusted models. Adding 3,4-DHBA, ribonic acid, and glycine to a traditional risk factor model improved the discrimination of hard microvascular events. CONCLUSIONS: While prospective studies directly assessing the predictive ability of these markers are needed, our results strengthen the role of clinical metabolomics in relation to risk assessment of diabetic complications in chronic type 1 diabetes.

Design and methodology of the PRIMETIME 1 cohort study: PRecIsion MEdicine based on kidney TIssue Molecular interrogation in diabetic nEphropathy.

Background: Clinical features of diabetic kidney disease alone cannot differentiate between the histopathology that defines diabetic nephropathy (DN) and non-diabetic nephropathy (NDN). A kidney biopsy is necessary to make the definitive diagnosis of DN. However, there is no consensus on when to perform a kidney biopsy in individuals with diabetes and kidney disease. Furthermore, the implications of NDN versus DN for management, morbidity and kidney prognosis are unclear. To address the gap in knowledge, we aimed to create a national retrospective cohort of people with diabetes and a performed kidney biopsy. Methods: Adults diagnosed with diabetes in Denmark between 1996 and 2020 who had a kidney biopsy performed were included. The cohort was established by linking a nationwide diabetes registry with the Danish Pathology Registry. Data from 11 national registries and databases were compiled. The type of kidney disease was classified using a three-step analysis of Systematized Nomenclature of Medicine codes reported in relation to the histopathological examinations of kidney tissue. The final cohort and classification of kidney disease was as follows: out of 485 989 individuals with diabetes 2586 were included, 2259 of whom had type 2 diabetes. We were able to classify 599 (26.5%) with DN, 703 (31.1%) with NDN and 165 (7.3%) with mixed disease in individuals with type 2 diabetes. In individuals with type 1 diabetes, 132 (40.4%) had DN, 73 (22.3%) NDN and 39 (11.9%) mixed disease. The remaining could not be classified or had normal histology. The overall median (Q1-Q3) follow-up time was 3.8 (1.6-7.2) years. Conclusions: This cohort is a novel platform based on high-quality registry data for important longitudinal studies of the impact of kidney disease diagnosis on prognosis. With regular updates of data from the Danish registries, the presented follow-up will increase over time and is only limited by emigration or death.

Functional Roles of N-Terminal Domains in Pullulanase from Human Gut Lactobacillus acidophilus.

Pullulanases are multidomain α-glucan debranching enzymes with one or more N-terminal domains (NTDs) including carbohydrate-binding modules (CBMs) and domains of unknown function (DUFs). To elucidate the roles of NTDs in Lactobacillus acidophilus NCFM pullulanase (LaPul), two truncated variants, Δ41-LaPul (lacking CBM41) and Δ(41+DUFs)-LaPul (lacking CBM41 and two DUFs), were produced recombinantly. LaPul recognized 1.3- and 2.2-fold more enzyme attack-sites on starch granules compared to Δ41-LaPul and Δ(41+DUFs)-LaPul, respectively, as measured by interfacial kinetics. Δ41-LaPul displayed markedly lower affinity for starch granules and ß-cyclodextrin (10- and >21-fold, respectively) in comparison to LaPul, showing substrate binding mainly stems from CBM41. Δ(41+DUFs)-LaPul exhibited a 12 °C lower melting temperature than LaPul and Δ41-LaPul, indicating that the DUFs are critical for LaPul stability. Notably, Δ41-LaPul exhibited a 14-fold higher turnover number (kcat) and 9-fold higher Michaelis constant (KM) compared to LaPul, while Δ(41+DUFs)-LaPul's values were close to those of LaPul, possibly due to the exposure of aromatic by truncation.

No Detectable Coagulation Activation After Vitamin K (MK-7) Supplementation in Patients on Dialysis With Functional Vitamin K Deficiency: A One-Year Randomized, Placebo-Controlled Study.

OBJECTIVE: Patients on dialysis treatment have poor functional vitamin K status, and this may increase the risk of vascular calcification. Vitamin K supplementation may therefore be relevant in patients on dialysis, but the procoagulant effects have not been studied. We evaluated effects of menaquinone-7 (MK-7) supplementation on biomarkers of coagulation in patients on dialysis. METHODS: Double-blinded, placebo-controlled study in 123 patients on dialysis randomized to 52 weeks of vitamin K (MK-7, 360 µg/daily, n = 61) or placebo (n = 62). Measurements at baseline and after 52 weeks of intervention included thrombin generation (endogenous thrombin potential, peak thrombin concentration, time to peak, and lag time); clot activities of vitamin K-dependent coagulation factors (F) II, VII, IX, and X; prothrombin fragment 1 + 2 (F1+2); and proteins induced by vitamin K absence II (PIVKA-II). Between-group differences (vitamin K vs. placebo) at 52 weeks were determined with an analysis of covariance. Within-group changes in vitamin K and placebo groups were analyzed with a paired t-test. Vascular adverse events and serious adverse events were registered based on hospital records, laboratory data, and participant interviews and compared between groups using Fisher's exact test or Pearson's Chi-Squared test. RESULTS: A between-group difference at 52 weeks was observed for PIVKA-II (P < .001). PIVKA-II decreased significantly from baseline to 52 weeks in the vitamin K group, but not in the placebo group. We observed no between-group differences or within-group changes for biomarkers of coagulation, except for FVII clot activity which was reduced in the placebo group (P = .04), and no between-group differences in adverse events and serious adverse events. CONCLUSION: One year of vitamin K supplementation in patients on dialysis has no detectable effects on biomarkers of coagulation activation, clot activities of vitamin K-dependent coagulation factors, and vascular events or death, indicating no procoagulant effects of this treatment.

Precision Medicine and/or Biomarker Based Therapy in T2DM: Ready for Prime Time?

Approximately 30-40% of people with type 2 diabetes mellitus develop chronic kidney disease. This is characterised by elevated blood pressure, declining kidney function and enhanced cardiovascular morbidity and mortality. Increased albuminuria and decreasing estimated glomerular function has to be evaluated regularly to diagsnose kidney disease. New biomarkers may facilitate early diagnosis and provide infomation on undlying pathology thereby supporting early precision intervention for the optimal benefit. A number of biomarkers have been suggested but are not yet implemented in clinical practice. iI the future such bimarkers may pave the way for personalized treatment.

Rationale and design of a prospective, clinical study of kidney biopsies in people with type 2 diabetes and severely increased albuminuria (the PRIMETIME 2 study).

INTRODUCTION: Diabetic kidney disease is a severe complication of diabetes. The diagnosis is based on clinical characteristics such as persistently elevated albuminuria, hypertension and decline in kidney function, although this definition is not specific to kidney disease caused by diabetes. The only way to establish an accurate diagnosis-diabetic nephropathy-is by performing a kidney biopsy. The histological presentation of diabetic nephropathy can be associated with a heterogeneous range of histological features with many pathophysiological factors involved demonstrating the complexity of the condition. Current treatment strategies aim to slow disease progression and are not specific to the underlying pathological processes.This study will investigate the prevalence of diabetic nephropathy in individuals with type 2 diabetes (T2D) and severely elevated albuminuria. The deep molecular characterisation of the kidney biopsy and biological specimens may pave the way for improved diagnostic accuracy and a better understanding of the pathological processes involved and may also reveal new targets for individualised treatment. METHODS AND ANALYSIS: In the PRecIsion MEdicine based on kidney TIssue Molecular interrogation in diabetic nEphropathy 2 study, research kidney biopsies will be performed in 300 participants with T2D, urine albumin/creatinine ratio ≥700 mg/g and estimated glomerular filtration ratio >30 mL/min/1.73 m2. Cutting-edge molecular technologies will be applied to the kidney, blood, urine, faeces and saliva samples for comprehensive multi-omics profiling. The associated disease course and clinical outcomes will be assessed by annual follow-up for 20 years. ETHICS AND DISSEMINATION: The Danish Regional Committee on Health Research Ethics and the Knowledge Center on Data Protection (in the Capital Region of Denmark) have granted approval for the study. The results will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04916132.

Improved Hydrolysis of Granular Starches by a Psychrophilic α-Amylase Starch Binding Domain-Fusion.

Degradation of starch granules by a psychrophilic α-amylase, AHA, from the Antarctic bacterium Pseudoalteromonas haloplanktis TAB23 was facilitated by C-terminal fusion to a starch-binding domain (SBD) from either Aspergillus niger glucoamylase (SBDGA) or Arabidopsis thaliana glucan, water dikinase 3 (SBDGWD3) via a decapeptide linker. Depending on the waxy, normal or high-amylose starch type and the botanical source, the AHA-SBD fusion enzymes showed up to 3 times higher activity than AHA wild-type. The SBD-fusion thus increased the density of enzyme attack-sites and binding-sites on the starch granules by up to 5- and 7-fold, respectively, as measured using an interfacial catalysis approach that combined conventional Michaelis-Menten kinetics, with the substrate in excess, and inverse kinetics, having enzyme in excess, with enzyme-starch granule adsorption isotherms. Higher substrate affinity of the SBDGA compared to SBDGWD3 was accompanied by the superior activity of AHA-SBDGA in agreement with the Sabatier principle of adsorption limited heterogenous catalysis.

Surface Plasmon Resonance Analysis for Quantifying Protein-Carbohydrate Interactions.

During the past two decades, surface plasmon resonance (SPR) analysis has emerged as an important tool for studying protein-carbohydrate interactions, with several commercial instruments available. Binding affinities in the nM to mM range can be determined; however, there are pitfalls that require careful experimental design to avoid. Here we give an overview of each step in the SPR analysis from immobilization to data analysis, providing key points of consideration that will allow practitioners to achieve reliable and reproducible results.

Interfacial Catalysis during Amylolytic Degradation of Starch Granules: Current Understanding and Kinetic Approaches.

Enzymatic hydrolysis of starch granules forms the fundamental basis of how nature degrades starch in plant cells, how starch is utilized as an energy resource in foods, and develops efficient, low-cost saccharification of starch, such as bioethanol and sweeteners. However, most investigations on starch hydrolysis have focused on its rates of degradation, either in its gelatinized or soluble state. These systems are inherently more well-defined, and kinetic parameters can be readily derived for different hydrolytic enzymes and starch molecular structures. Conversely, hydrolysis is notably slower for solid substrates, such as starch granules, and the kinetics are more complex. The main problems include that the surface of the substrate is multifaceted, its chemical and physical properties are ill-defined, and it also continuously changes as the hydrolysis proceeds. Hence, methods need to be developed for analyzing such heterogeneous catalytic systems. Most data on starch granule degradation are obtained on a long-term enzyme-action basis from which initial rates cannot be derived. In this review, we discuss these various aspects and future possibilities for developing experimental procedures to describe and understand interfacial enzyme hydrolysis of native starch granules more accurately.

Effects of two- and twelve-weeks sodium-glucose cotransporter 2 inhibition on DNA and RNA oxidation: two randomized, placebo-controlled trials.

Animal studies have shown that SGLT2 inhibition decreases oxidative stress, which may explain the cardiovascular protective effects observed following SGLT2 inhibition treatment. Thus, we investigated the effects of two and twelve weeks SGLT2 inhibition on DNA and RNA oxidation. Individuals with type 2 diabetes (n = 31) were randomized to two weeks of treatment with the SGLT2 inhibitor empagliflozin treatment (25 mg once daily) or placebo. The primary outcome was changes in DNA and RNA oxidation measured as urinary excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo), respectively. In another trial, individuals with type 2 diabetes (n = 35) were randomized to twelve weeks of dapagliflozin treatment (10 mg once daily) or placebo in a crossover study. Changes in urinary excretion of 8-oxodG and 8-oxoGuo were investigated as a posthoc analysis. Compared with placebo treatment, two weeks of empagliflozin treatment did not change urinary excretion of 8-oxodG (between-group difference: 0.3 nmol/24-hour (95% CI: -4.2 to 4.8)) or 8-oxoGuo (1.3 nmol/24-hour (95% CI: -4.7 to 7.3)). From a mean baseline 8-oxodG/creatinine urinary excretion of 1.34 nmol/mmol, dapagliflozin-treated individuals changed 8-oxodG/creatinine by -0.17 nmol/mmol (95% CI: -0.29 to -0.04) following twelve weeks of treatment, whereas placebo-treated individuals did not change 8-oxodG/creatinine (within-group effect: 0.10 nmol/mmol (95% CI: -0.02 to 0.22)) resulting in a significant between-group difference (p = 0.01). Urinary excretion of 8-oxoGuo was unaffected by dapagliflozin treatment. In conclusion, two weeks of empagliflozin treatment did not change DNA or RNA oxidation. However, a posthoc analysis revealed that longer-term dapagliflozin treatment decreased DNA oxidation. Clinicaltrials.gov: NCT02890745 and NCT02914691.HighlightsPlasma ferritin correlated with DNA and RNA oxidation in individuals with T2D.Twelve weeks dapagliflozin treatment decreased DNA oxidation.Dapagliflozin and empagliflozin treatment did not change RNA oxidation.Lipid peroxidation was unaffected by two weeks empagliflozin treatment.

Exploration of the Transglycosylation Activity of Barley Limit Dextrinase for Production of Novel Glycoconjugates.

A few α-glucan debranching enzymes (DBEs) of the large glycoside hydrolase family 13 (GH13), also known as the α-amylase family, have been shown to catalyze transglycosylation as well as hydrolysis. However, little is known about their acceptor and donor preferences. Here, a DBE from barley, limit dextrinase (HvLD), is used as a case study. Its transglycosylation activity is studied using two approaches; (i) natural substrates as donors and different p-nitrophenyl (pNP) sugars as well as different small glycosides as acceptors, and (ii) α-maltosyl and α-maltotriosyl fluorides as donors with linear maltooligosaccharides, cyclodextrins, and GH inhibitors as acceptors. HvLD showed a clear preference for pNP maltoside both as acceptor/donor and acceptor with the natural substrate pullulan or a pullulan fragment as donor. Maltose was the best acceptor with α-maltosyl fluoride as donor. The findings highlight the importance of the subsite +2 of HvLD for activity and selectivity when maltooligosaccharides function as acceptors. However, remarkably, HvLD is not very selective when it comes to aglycone moiety; different aromatic ring-containing molecules besides pNP could function as acceptors. The transglycosylation activity of HvLD can provide glycoconjugate compounds with novel glycosylation patterns from natural donors such as pullulan, although the reaction would benefit from optimization.

Study protocol of the InterVitaminK trial: a Danish population-based randomised double-blinded placebo-controlled trial of the effects of vitamin K (menaquinone-7) supplementation on cardiovascular, metabolic and bone health.

INTRODUCTION: Vitamin K has been suggested to have protective effects against progression of vascular calcification and development of cardiovascular disease (CVD). However, few well-powered randomised controlled trials have examined whether vitamin K prevents progression of vascular calcification in individuals from the general population. The aim of the InterVitaminK trial is to investigate the effects of vitamin K supplementation (menaquinone-7, MK-7) on cardiovascular, metabolic, respiratory and bone health in a general ageing population with detectable vascular calcification. METHODS AND ANALYSIS: The InterVitaminK trial is a randomised, double-blinded, placebo-controlled, trial. A total of 450 men and women aged 52-82 years with detectable coronary artery calcification (CAC), but without manifest CVD, will be randomised (1:1) to receive daily MK-7 (333 µg/day) or placebo tablets for 3 years. Health examinations are scheduled at baseline, and after 1, 2 and 3 years of intervention. Health examinations include cardiac CT scans, measurements of arterial stiffness, blood pressure, lung function, physical function, muscle strength, anthropometric measures, questionnaires on general health and dietary intake, and blood and urine sampling. The primary outcome is progression of CAC from baseline to 3-year follow-up. The trial has 89% power to detect a between-group difference of at least 15%. Secondary outcomes are bone mineral density, pulmonary function and biomarkers of insulin resistance. ETHICS AND DISSEMINATION: Oral MK-7 supplementation is considered safe and has not been found to cause severe adverse events. The Ethical Committee of the Capital Region (H-21033114) approved the protocol. Written informed consent is obtained from all participants and the trial is conducted in accordance with the Declaration of Helsinki II. Both negative and positive findings will be reported. TRIAL REGISTRATION NUMBER: NCT05259046.

Structural and functional characterization of a multi-domain GH92 α-1,2-mannosidase from Neobacillus novalis.

Many secreted eukaryotic proteins are N-glycosylated with oligosaccharides composed of a high-mannose N-glycan core and, in the specific case of yeast cell-wall proteins, an extended α-1,6-mannan backbone carrying a number of α-1,2- and α-1,3-mannose substituents of varying lengths. α-Mannosidases from CAZy family GH92 release terminal mannose residues from these N-glycans, providing access for the α-endomannanases, which then degrade the α-mannan backbone. Most characterized GH92 α-mannosidases consist of a single catalytic domain, while a few have extra domains including putative carbohydrate-binding modules (CBMs). To date, neither the function nor the structure of a multi-domain GH92 α-mannosidase CBM has been characterized. Here, the biochemical investigation and crystal structure of the full-length five-domain GH92 α-1,2-mannosidase from Neobacillus novalis (NnGH92) with mannoimidazole bound in the active site and an additional mannoimidazole bound to the N-terminal CBM32 are reported. The structure of the catalytic domain is very similar to that reported for the GH92 α-mannosidase Bt3990 from Bacteroides thetaiotaomicron, with the substrate-binding site being highly conserved. The function of the CBM32s and other NnGH92 domains was investigated by their sequential deletion and suggested that whilst their binding to the catalytic domain was crucial for the overall structural integrity of the enzyme, they appear to have little impact on the binding affinity to the yeast α-mannan substrate. These new findings provide a better understanding of how to select and optimize other multi-domain bacterial GH92 α-mannosidases for the degradation of yeast α-mannan or mannose-rich glycans.

The association between blood oxygen saturation and baroreflex sensitivity in adults with type 1 diabetes with and without albuminuria.

BACKGROUND: Low baroreflex sensitivity is an indicator of early cardiovascular autonomic neuropathy. We explored the association between baroreflex sensivity and blood oxygen saturation (SpO2) in type 1 diabetes and various degrees of microvascular disease. METHODS: In this Danish-Finnish cross-sectional multicentre study, baroreflex sensivity and SpO2 (pulse oximetry) were examined in persons with type 1 diabetes and normoalbuminuria (n = 98), microalbuminuria (n = 28), or macroalbuminuria (n = 43), and in non-diabetic controls (n = 54). Associations and differences between groups were analysed using regression models and adjustment included age, sex, smoking, HbA1c, blood haemoglobin, urine albumin creatinine ratio, body mass index, and estimated glomerular filtration rate. RESULTS: In type 1 diabetes, higher baroreflex sensitivity was associated with higher SpO2 before adjustment (% increase per one % increase in SpO2 = 20 % (95%CI: 11-30); p < 0.001) and the association remained significant after adjustment (p = 0.02). Baroreflex sensitivity was not different between non-diabetic controls and persons with type 1 diabetes and normoalbuminuria (p = 0.052). Compared with type 1 diabetes and normoalbuminuria, baroreflex sensitivity was lower in micro- (p < 0.001) and macroalbuminuria (p < 0.001). SpO2 was lower in persons with type 1 diabetes and normoalbuminuria compared with non-diabetic controls (p < 0.01). Within the participants with type 1 diabetes, SpO2 was not different in micro- or macroalbuminuria compared with normoalbuminuria (p-values > 0.05), but lower in macro-compared with microalbuminuria (p < 0.01). CONCLUSIONS: Lower baroreflex sensitivity was associated with lower SpO2 in type 1 diabetes. The present study support the hypothesis that hypoxia could be a therapeutic target in persons with type 1 diabetes.

Marker for kidney fibrosis is associated with inflammation and deterioration of kidney function in people with type 2 diabetes and microalbuminuria.

BACKGROUND: Diabetic kidney disease is a major cause of morbidity and mortality. Dysregulated turnover of collagen type III is associated with development of kidney fibrosis. We investigated whether a degradation product of collagen type III (C3M) was a risk marker for progression of chronic kidney disease (CKD), occurrence of cardiovascular disease (CVD), and mortality during follow up in people with type 2 diabetes (T2D) and microalbuminuria. Moreover, we investigated whether C3M was correlated with markers of inflammation and endothelial dysfunction at baseline. METHODS: C3M was measured in serum (sC3M) and urine (uC3M) in 200 participants with T2D and microalbuminuria included in an observational, prospective study at Steno Diabetes Center Copenhagen in Denmark from 2007-2008. Baseline measurements included 12 markers of inflammation and endothelial dysfunction. The endpoints were CVD, mortality, and CKD progression (>30% decline in eGFR). RESULTS: Mean (SD) age was 59 (9) years, eGFR 90 (17) ml/min/1.73m2 and median (IQR) urine albumin excretion rate 102 (39-229) mg/24-h. At baseline all markers for inflammation were positively correlated with sC3M (p≤0.034). Some, but not all, markers for endothelial dysfunction were correlated with C3M. Median follow-up ranged from 4.9 to 6.3 years. Higher sC3M was associated with CKD progression (with mortality as competing risk) with a hazard ratio (per doubling) of 2.98 (95% CI: 1.41-6.26; p = 0.004) adjusted for traditional risk factors. uC3M was not associated with CKD progression. Neither sC3M or uC3M were associated with risk of CVD or mortality. CONCLUSIONS: Higher sC3M was a risk factor for chronic kidney disease progression and was correlated with markers of inflammation.

Protein-plastic interactions: The driving forces behind the high affinity of a carbohydrate-binding module for polyethylene terephthalate.

Polyethylene terephthalate (PET) waste is a common pollutant in the environment, mainly due to resistance of the plastic to bio-degradation. Nevertheless, hydrolytic enzymes have been identified with activity on this substrate, which are continually being engineered to increase activity. Some insoluble biological polymers are degraded by enzymes with a multi-domain architecture, comprising of a catalytic domain, and a substrate-binding domain, such as a carbohydrate-binding module (CBM). Enzymes that degrade PET have been shown to have a higher activity when fused with these CBMs, indicating a promising route for engineering better enzymes for plastic bioprocessing. However, no detailed study of the affinity and binding mechanism of these domains on PET has yet been made. Here, we perform an in depth analysis of a binding domain from CBM family 2 on PET, showing that the affinity of the protein for the plastic is highly dependent on temperature and crystallinity of the plastic. We also investigate the mechanism of the interaction, and show how affinity may be engineered in both directions. CBM affinity for other synthetic polymers is also demonstrated for the first time. Our results demonstrate that the substrate affinity of fusion enzymes with binding modules can be tuned to the desired level.