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1.  Insulin resistance and hyperinsulinaemia in diabetic cardiomyopathy 
Nature reviews. Endocrinology  2015;12(3):144-153.
Insulin resistance, type 2 diabetes mellitus and associated hyperinsulinaemia can promote the development of a specific form of cardiomyopathy that is independent of coronary artery disease and hypertension. Termed diabetic cardiomyopathy, this form of cardiomyopathy is a major cause of morbidity and mortality in developed nations, and the prevalence of this condition is rising in parallel with increases in the incidence of obesity and type 2 diabetes mellitus. Of note, female patients seem to be particularly susceptible to the development of this complication of metabolic disease. The diabetic cardiomyopathy observed in insulin-resistant or hyperinsulinaemic states is characterized by impaired myocardial insulin signalling, mitochondrial dysfunction, endoplasmic reticulum stress, impaired calcium homeostasis, abnormal coronary microcirculation, activation of the sympathetic nervous system, activation of the renin–angiotensin–aldosterone system and maladaptive immune responses. These pathophysiological changes result in oxidative stress, fibrosis, hypertrophy, cardiac diastolic dysfunction and eventually systolic heart failure. This Review highlights a surge in diabetic cardiomyopathy research, summarizes current understanding of the molecular mechanisms underpinning this condition and explores potential preventive and therapeutic strategies.
PMCID: PMC4753054  PMID: 26678809
2.  Uric Acid Promotes Left Ventricular Diastolic Dysfunction in Mice Fed a Western Diet 
Hypertension  2014;65(3):531-539.
The rising obesity rates parallel increased consumption of a western diet, high in fat and fructose, which is associated with increased uric acid. Population based data support that elevated serum uric acids are associated with left ventricular hypertrophy and diastolic dysfunction. However, the mechanism by which excess uric acid promotes these maladaptive cardiac effects has not been explored. In assessing the role of western diet-induced increases in uric acid, we hypothesized that reductions in uric acid would prevent western diet-induced development of cardiomyocyte hypertrophy, cardiac stiffness and impaired diastolic relaxation by reducing growth and pro-fibrotic signaling pathways. Four week-old C57BL6/J male mice were fed excess fat (46%) and fructose (17.5%) with or without allopurinol (125mg/L), a xanthine oxidase inhibitor, for 16 weeks. The western diet induced increases in serum uric acid along with increases in cardiac tissue xanthine oxidase activity temporally related to increases in body weight, fat mass, and insulin resistance without changes in blood pressure. The western diet induced cardiomyocte hypertrophy, myocardial oxidative stress, interstitial fibrosis, and impaired diastolic relaxation. Further, the western diet enhanced activation of the S6 kinase-1 growth pathway and the pro-fibrotic transforming growth factor (TGF)-β1/Smad2/3 signaling pathway, and macrophage pro-inflammatory polarization. All results improved with allopurinol treatment, which lowered cardiac xanthine oxidase as well as serum uric acid levels. These findings support the notion that increased production of uric acid with intake of a western diet, promotes cardiomyocyte hypertrophy, inflammation and oxidative stress that lead to myocardial fibrosis and associated impaired diastolic relaxation.
PMCID: PMC4370431  PMID: 25489061
Obesity; Inflammation; Uric Acid; Cardiac Remodeling; Oxidant Stress; Macrophage Polarization
3.  Genome mapping of postzygotic hybrid necrosis in an interspecific pear population 
Horticulture Research  2016;3:15064-.
Deleterious epistatic interactions in plant inter- and intraspecific hybrids can cause a phenomenon known as hybrid necrosis, characterized by a typical seedling phenotype whose main distinguishing features are dwarfism, tissue necrosis and in some cases lethality. Identification of the chromosome regions associated with this type of incompatibility is important not only to increase our understanding of the evolutionary diversification that led to speciation but also for breeding purposes. Development of molecular markers linked to the lethal genes will allow breeders to avoid incompatible inbred combinations that could affect the expression of important agronomic tratis co-segregating with these genes. Although hybrid necrosis has been reported in several plant taxa, including Rosaceae species, this phenomenon has not been described previously in pear. In the interspecific pear population resulting from a cross between PEAR3 (Pyrus bretschneideri × Pyrus communis) and ‘Moonglow’ (P. communis), we observed two types of hybrid necrosis, expressed at different stages of plant development. Using a combination of previously mapped and newly developed genetic markers, we identified three chromosome regions associated with these two types of lethality, which were genetically independent. One type resulted from a negative epistatic interaction between a locus on linkage group 5 (LG5) of PEAR3 and a locus on LG1 of ‘Moonglow’, while the second type was due to a gene that maps to LG2 of PEAR3 and which either acts alone or more probably interacts with another gene of unknown location inherited from ‘Moonglow’.
PMCID: PMC4702180  PMID: 26770810
4.  Enzymatic cellulose oxidation is linked to lignin by long-range electron transfer 
Scientific Reports  2015;5:18561.
Enzymatic oxidation of cell wall polysaccharides by lytic polysaccharide monooxygenases (LPMOs) plays a pivotal role in the degradation of plant biomass. While experiments have shown that LPMOs are copper dependent enzymes requiring an electron donor, the mechanism and origin of the electron supply in biological systems are only partly understood. We show here that insoluble high molecular weight lignin functions as a reservoir of electrons facilitating LPMO activity. The electrons are donated to the enzyme by long-range electron transfer involving soluble low molecular weight lignins present in plant cell walls. Electron transfer was confirmed by electron paramagnetic resonance spectroscopy showing that LPMO activity on cellulose changes the level of unpaired electrons in the lignin. The discovery of a long-range electron transfer mechanism links the biodegradation of cellulose and lignin and sheds new light on how oxidative enzymes present in plant degraders may act in concert.
PMCID: PMC4685257  PMID: 26686263
5.  Harnessing the potential of LPMO-containing cellulase cocktails poses new demands on processing conditions 
The emerging bioeconomy depends on improved methods for processing of lignocellulosic biomass to fuels and chemicals. Saccharification of lignocellulose to fermentable sugars is a key step in this regard where enzymatic catalysis plays an important role and is a major cost driver. Traditionally, enzyme cocktails for the conversion of cellulose to fermentable sugars mainly consisted of hydrolytic cellulases. However, the recent discovery of lytic polysaccharide monooxygenases (LPMOs), which cleave cellulose using molecular oxygen and an electron donor, has provided new tools for biomass saccharification.
Current commercial enzyme cocktails contain LPMOs, which, considering the unique properties of these enzymes, may change optimal processing conditions. Here, we show that such modern cellulase cocktails release up to 60 % more glucose from a pretreated lignocellulosic substrate under aerobic conditions compared to anaerobic conditions. This higher yield correlates with the accumulation of oxidized products, which is a signature of LPMO activity. Spiking traditional cellulase cocktails with LPMOs led to increased saccharification yields, but only under aerobic conditions. LPMO activity on pure cellulose depended on the addition of an external electron donor, whereas this was not required for LPMO activity on lignocellulose.
In this study, we demonstrate a direct correlation between saccharification yield and LPMO activity of commercial enzyme cocktails. Importantly, we show that the LPMO contribution to overall efficiency may be large if process conditions are adapted to the key determinants of LPMO activity, namely the presence of electron donors and molecular oxygen. Thus, the advent of LPMOs has a great potential, but requires rethinking of industrial bioprocessing procedures.
Electronic supplementary material
The online version of this article (doi:10.1186/s13068-015-0376-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4659242  PMID: 26609322
GH61; Biorefinery; Cellulase; AA9; Lignin; Bioeconomy; Biofuel
7.  Lactobacillus plantarum displaying CCL3 chemokine in fusion with HIV-1 Gag derived antigen causes increased recruitment of T cells 
Chemokines are attractive candidates for vaccine adjuvants due to their ability to recruit the immune cells. Lactic acid bacteria (LAB)-based delivery vehicles have potential to be used as a cheap and safe option for vaccination. Chemokine produced on the surface of LAB may potentially enhance the immune response to an antigen and this approach can be considered in development of future mucosal vaccines.
We have constructed strains of Lactobacillusplantarum displaying a chemokine on their surface. L. plantarum was genetically engineered to express and anchor to the surface a protein called CCL3Gag. CCL3Gag is a fusion protein comprising of truncated HIV-1 Gag antigen and the murine chemokine CCL3, also known as MIP-1α. Various surface anchoring strategies were explored: (1) a lipobox-based covalent membrane anchor, (2) sortase-mediated covalent cell wall anchoring, (3) LysM-based non-covalent cell wall anchoring, and (4) an N-terminal signal peptide-based transmembrane anchor. Protein production and correct localization were confirmed using Western blotting, flow cytometry and immunofluorescence microscopy. Using a chemotaxis assay, we demonstrated that CCL3Gag-producing L. plantarum strains are able to recruit immune cells in vitro.
The results show the ability of engineered L. plantarum to produce a functional chemotactic protein immobilized on the bacterial surface. We observed that the activity of surface-displayed CCL3Gag differed depending on the type of anchor used. The chemokine which is a part of the bacteria-based vaccine may increase the recruitment of immune cells and, thereby, enhance the reaction of the immune system to the vaccine.
Electronic supplementary material
The online version of this article (doi:10.1186/s12934-015-0360-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4618854  PMID: 26494531
Chemokine; CCL3; MIP-1α; Chemotaxis; HIV; Lactic acid bacteria; Lactobacillus; Mucosal vaccine
8.  Characterization of microbial community structure during continuous anaerobic digestion of straw and cow manure 
Microbial Biotechnology  2015;8(5):815-827.
Responses of bacterial and archaeal communities to the addition of straw during anaerobic digestion of manure at different temperatures (37°C, 44°C and 52°C) were investigated using five laboratory-scale semi-continuous stirred tank reactors. The results revealed that including straw as co-substrate decreased the species richness for bacteria, whereas increasing the operating temperature decreased the species richness for both archaea and bacteria, and also the evenness of the bacteria. Taxonomic classifications of the archaeal community showed that Methanobrevibacter dominated in the manure samples, while Methanosarcina dominated in all digesters regardless of substrate. Increase of the operating temperature to 52°C led to increased relative abundance of Methanoculleus and Methanobacterium. Among the bacteria, the phyla Firmicutes and Bacteroidetes dominated within all samples. Compared with manure itself, digestion of manure resulted in a higher abundance of an uncultured class WWE1 and lower abundance of Bacilli. Adding straw to the digesters increased the level of Bacteroidia, while increasing the operating temperature decreased the level of this class and instead increased the relative abundance of an uncultured genus affiliated to order MBA08 (Clostridia). A considerable fraction of bacterial sequences could not be allocated to genus level, indicating that novel phylotypes are resident in these communities.
PMCID: PMC4554469  PMID: 26152665
9.  Vascular stiffness in insulin resistance and obesity 
Obesity, insulin resistance, and type 2 diabetes are associated with a substantially increased prevalence of vascular fibrosis and stiffness, with attendant increased risk of cardiovascular and chronic kidney disease. Although the underlying mechanisms and mediators of vascular stiffness are not well understood, accumulating evidence supports the role of metabolic and immune dysregulation related to increased adiposity, activation of the renin angiotensin aldosterone system, reduced bioavailable nitric oxide, increased vascular extracellular matrix (ECM) and ECM remodeling in the pathogenesis of vascular stiffness. This review will give a brief overview of the relationship between obesity, insulin resistance and increased vascular stiffness to provide a contemporary understanding of the proposed underlying mechanisms and potential therapeutic strategies.
PMCID: PMC4536384  PMID: 26321962
obesity; insulin resistance; vascular resistance; cardiovascular disease
10.  The effect of short-term high versus normal protein intake on whole-body protein synthesis and balance in children following cardiac surgery: a randomized double-blind controlled clinical trial 
Nutrition Journal  2015;14:72.
Infants undergoing cardiac surgery are at risk of a negative protein balance, due to increased proteolysis in response to surgery and the cardiopulmonary bypass circuit, and limited intake. The aim of the study was to quantify the effect on protein kinetics of a short-term high-protein (HP) diet in infants following cardiac surgery.
In a prospective, double-blinded, randomized trial we compared the effects of a HP (5 g · kg−1 · d−1) versus normal protein (NP, 2 g · kg−1 · d−1) enteral diet on protein kinetics in children <24 months, on day 2 following surgical repair of congenital heart disease. Valine kinetics and fractional albumin synthesis rate (FSRalb) were measured with mass spectrometry using [1-13C]valine infusion. The Mann–Whitney U test was used to investigate differences between group medians. Additionally, the Hodges-Lehmann procedure was used to create a confidence interval with a point estimate of median differences between groups.
Twenty-eight children (median age 9 months, median weight 7 kg) participated in the study, of whom in only 20 subjects isotopic data could be used for final calculations. Due to underpowering of our study, we could not draw conclusions on the primary outcome parameters. We observed valine synthesis rate of 2.73 (range: 0.94 to 3.36) and 2.26 (1.85 to 2.73) μmol · kg−1 · min−1 in the HP and NP diet, respectively. The net valine balance was 0.54 (−0.73 to 1.75) and 0.24 (−0.20 to 0.63) μmol · kg−1 · min−1 in the HP and NP group. Between groups, there was no difference in FSRalb. We observed increased oxidation and BUN in the HP diet, compared to the NP diet, as a plausible explanation of the metabolic fate of surplus protein.
It is plausible that the surplus protein in the HP group has caused the increase of valine oxidation and ureagenesis, compared to the NP group. Because too few patients had completed the study, we were unable to draw conclusions on the effect of a HP diet on protein synthesis and balance. We present our results as new hypothesis generating data.
Trial registration
Dutch Trial Register NTR2334.
PMCID: PMC4517637  PMID: 26215396
Proteins; Isotopes; Child; Congenital heart defect; Intensive care
12.  Fine grained osseointegrative coating improves biocompatibility of PEEK in heterotopic sheep model 
Background and aim
Polyetheretherketone (PEEK) materials already have been used successfully in orthopedic and especially spine surgery. PEEK is radiolucent and comparable with bone regarding elasticity. However, PEEK is inert and the adhesion of PEEK implants to bone tissue proceeds slowly because of their relatively low biocompatibility. The aim of the study is to evaluate the effect of titanium and CaP coating on the adhesion of bone tissue.
Material and Methods
Six adult sheep (body weight 57.6 ± 3.9 kg) were included in this study. Three different types of cylindrical dowels (12 mm length x 8 mm diameter) were implanted in long bones (tibia and femur): PEEK dowels without coating (the control group), and PEEK dowels with a nanocoating of calcium phosphate (CaP group) or titanium (titanium group). Animals were sacrificed after 6, 12 and 26 weeks. Dowels were explanted for micro CT and histology.
Bone implant contact (BIC) ratio was significantly higher in the titanium versus control groups in the 6 to 12 weeks period (p = 0.03). The ratio between bone volume and tissue volume (BV/TV) was significantly higher in titanium versus control in the 6 to 12 weeks period (p = 0.02). A significant correlation between BIC and BV/TV was seen (r = 0.85, p < 0.05).
Coating of PEEK dowels with a nanocoating of titanium has beneficial effects on adhesion of bone tissue.
PMCID: PMC4528570  PMID: 26273553
titanium; Bone ingrowth; Polyetheretherketone; spinal surgery; Nanotopography; In vivo test
13.  Surface display of an anti-DEC-205 single chain Fv fragment in Lactobacillus plantarum increases internalization and plasmid transfer to dendritic cells in vitro and in vivo 
Lactic acid bacteria (LAB) are promising vehicles for delivery of a variety of medicinal compounds, including antigens and cytokines. It has also been established that LAB are able to deliver cDNA to host cells. To increase the efficiency of LAB-driven DNA delivery we have constructed Lactobacillus plantarum strains targeting DEC-205, which is a receptor located at the surface of dendritic cells (DCs). The purpose was to increase uptake of bacterial cells, which could lead to improved cDNA delivery to immune cells.
Anti-DEC-205 antibody (aDec) was displayed at the surface of L. plantarum using three different anchoring strategies: (1) covalent anchoring of aDec to the cell membrane (Lipobox domain, Lip); (2) covalent anchoring to the cell wall (LPXTG domain, CWA); (3) non-covalent anchoring to the cell wall (LysM domain, LysM). aDec was successfully expressed in all three strains, but surface location of the antibody could only be demonstrated for the two strains with cell wall anchors (CWA and LysM). Co-incubation of the engineered strains and DCs showed increased uptake when anchoring aDec using the CWA or LysM anchors. In a competition assay, free anti-DEC abolished the increased uptake, showing that the internalization is due to specific interactions between the DEC-205 receptor and aDec. To test plasmid transfer, a plasmid for expression of GFP under control of an eukaryotic promoter was transformed into the aDec expressing strains and GFP expression in DCs was indeed increased when using the strains producing cell-wall anchored aDec. Plasmid transfer to DCs in the gastro intestinal tract was also detected using a mouse model. Surprisingly, in mice the highest expression of GFP was observed for the strain in which aDec was coupled to the cell membrane.
The results show that surface expression of aDec leads to increased internalization of L. plantarum and plasmid transfer in DCs and that efficiency depends on the type of anchor used. Interestingly, in vitro data indicates that cell wall anchoring is more effective, whereas in vivo data seem to indicate that anchoring to the cell membrane is preferable. It is likely that the more embedded localization of aDec in the latter case is favorable when cells are exposed to the harsh conditions of the gastro-intestinal tract.
PMCID: PMC4491208  PMID: 26141059
Dendritic cell; DEC-205; Lactic acid bacteria; Drug delivery; Cellular targeting; Surface display
14.  Autoantibody-Targeted Treatments for Acute Exacerbations of Idiopathic Pulmonary Fibrosis 
PLoS ONE  2015;10(6):e0127771.
Severe acute exacerbations (AE) of idiopathic pulmonary fibrosis (IPF) are medically untreatable and often fatal within days. Recent evidence suggests autoantibodies may be involved in IPF progression. Autoantibody-mediated lung diseases are typically refractory to glucocorticoids and nonspecific medications, but frequently respond to focused autoantibody reduction treatments. We conducted a pilot trial to test the hypothesis that autoantibody-targeted therapies may also benefit AE-IPF patients.
Eleven (11) critically-ill AE-IPF patients with no evidence of conventional autoimmune diseases were treated with therapeutic plasma exchanges (TPE) and rituximab, supplemented in later cases with intravenous immunoglobulin (IVIG). Plasma anti-epithelial (HEp-2) autoantibodies and matrix metalloproteinase-7 (MMP7) were evaluated by indirect immunofluorescence and ELISA, respectively. Outcomes among the trial subjects were compared to those of 20 historical control AE-IPF patients treated with conventional glucocorticoid therapy prior to this experimental trial.
Nine (9) trial subjects (82%) had improvements of pulmonary gas exchange after treatment, compared to one (5%) historical control. Two of the three trial subjects who relapsed after only five TPE responded again with additional TPE. The three latest subjects who responded to an augmented regimen of nine TPE plus rituximab plus IVIG have had sustained responses without relapses after 96-to-237 days. Anti-HEp-2 autoantibodies were present in trial subjects prior to therapy, and were reduced by TPE among those who responded to treatment. Conversely, plasma MMP7 levels were not systematically affected by therapy nor correlated with clinical responses. One-year survival of trial subjects was 46+15% vs. 0% among historical controls. No serious adverse events were attributable to the experimental medications.
This pilot trial indicates specific treatments that reduce autoantibodies might benefit some severely-ill AE-IPF patients. These findings have potential implications regarding mechanisms of IPF progression, and justify considerations for incremental trials of autoantibody-targeted therapies in AE-IPF patients.
Trial Registration NCT01266317
PMCID: PMC4470587  PMID: 26083430
15.  Perturbation training to promote safe independent mobility post-stroke: study protocol for a randomized controlled trial 
BMC Neurology  2015;15:87.
Falls are one of the most common medical complications post-stroke. Physical exercise, particularly exercise that challenges balance, reduces the risk of falls among healthy and frail older adults. However, exercise has not proven effective for preventing falls post-stroke. Falls ultimately occur when an individual fails to recover from a loss of balance. Thus, training to specifically improve reactive balance control could prevent falls. Perturbation training aims to improve reactive balance control by repeatedly exposing participants to postural perturbations. There is emerging evidence that perturbation training reduces fall rates among individuals with neurological conditions, such as Parkinson disease. The primary aim of this work is to determine if perturbation-based balance training can reduce occurrence of falls in daily life among individuals with chronic stroke. Secondary objectives are to determine the effect of perturbation training on balance confidence and activity restriction, and functional balance and mobility.
Individuals with chronic stroke will be recruited. Participants will be randomly assigned to one of two groups: 1) perturbation training, or 2) ‘traditional’ balance training. Perturbation training will involve both manual perturbations (e.g., a push or pull from a physiotherapist), and rapid voluntary movements to cause a loss of balance. Training will occur twice per week for 6 weeks. Participants will record falls and activity for 12 months following completion of the training program. Standardized clinical tools will be used to assess functional balance and mobility, and balance confidence before and after training.
Falls are a significant problem for those with stroke. Despite the large body of work demonstrating effective interventions, such as exercise, for preventing falls in other populations, there is little evidence for interventions that prevent falls post-stroke. The proposed study will investigate a novel and promising intervention: perturbation training. If effective, this training has the potential to not only prevent falls, but to also improve safe independent mobility and engagement in daily activities for those with stroke.
Trial registration
Current Controlled Trials: ISRCTN05434601.
PMCID: PMC4456796  PMID: 26048054
Stroke; Rehabilitation; Accidental falls; Postural balance
16.  Routes of Acquisition of the Gut Microbiota of the Honey Bee Apis mellifera 
Applied and Environmental Microbiology  2014;80(23):7378-7387.
Studies of newly emerged Apis mellifera worker bees have demonstrated that their guts are colonized by a consistent core microbiota within several days of eclosure. We conducted experiments aimed at illuminating the transmission routes and spatiotemporal colonization dynamics of this microbiota. Experimental groups of newly emerged workers were maintained in cup cages and exposed to different potential transmission sources. Colonization patterns were evaluated using quantitative real-time PCR (qPCR) to assess community sizes and using deep sequencing of 16S rRNA gene amplicons to assess community composition. In addition, we monitored the establishment of the ileum and rectum communities within workers sampled over time from natural hive conditions. The study verified that workers initially lack gut bacteria and gain large characteristic communities in the ileum and rectum within 4 to 6 days within hives. Typical communities, resembling those of workers within hives, were established in the presence of nurse workers or nurse worker fecal material, and atypical communities of noncore or highly skewed compositions were established when workers were exposed only to oral trophallaxis or hive components (comb, honey, bee bread). The core species of Gram-negative bacteria, Snodgrassella alvi, Gilliamella apicola, and Frischella perrara, were dependent on the presence of nurses or hindgut material, whereas some Gram-positive species were more often transferred through exposure to hive components. These results indicate aspects of the colony life cycle and behavior that are key to the propagation of the characteristic honey bee gut microbiota.
PMCID: PMC4249178  PMID: 25239900
17.  Proteomic Investigation of the Response of Enterococcus faecalis V583 when Cultivated in Urine 
PLoS ONE  2015;10(4):e0126694.
Enterococcus faecalis is a robust bacterium, which is able to survive in and adapt to hostile environments such as the urinary tract and bladder. In this label-free quantitative proteomic study based on MaxQuant LFQ algorithms, we identified 127 proteins present in the secretome of the clinical vancomycin-resistant isolate E. faecalis V583 and we compared proteins secreted in the initial phase of cultivation in urine with the secretome during cultivation in standard laboratory medium, 2xYT. Of the 54 identified proteins predicted to be secreted, six were exclusively found after cultivation in urine including the virulence factor EfaA (“endocarditis specific antigen”) and its homologue EF0577 (“adhesion lipoprotein”). These two proteins are both involved in manganese transport, known to be an important determinant of colonization and infection, and may additionally function as adhesins. Other detected urine-specific proteins are involved in peptide transport (EF0063 and EF3106) and protease inhibition (EF3054). In addition, we found an uncharacterized protein (EF0764), which had not previously been linked to the adaptation of V583 to a urine environment, and which is unique to E. faecalis. Proteins found in both environments included a histone-like protein, EF1550, that was up-regulated during cultivation in urine and that has a homologue in streptococci (HlpA) known to be involved in bacterial adhesion to host cells. Up-regulated secreted proteins included autolysins. These results from secretome analyses are largely compatible with previously published data from transcriptomics studies. All in all, the present data indicate that transport, in particular metal transport, adhesion, cell wall remodelling and the unknown function carried out by the unique EF0764 are important for enterococcal adaptation to the urine environment. These results provide a basis for a more targeted exploration of novel proteins involved in the adaptability and pathogenicity of E. faecalis.
PMCID: PMC4411035  PMID: 25915650
18.  A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes 
Enzymes that degrade or modify polysaccharides are widespread in pro- and eukaryotes and have multiple biological roles and biotechnological applications. Recent advances in genome and secretome sequencing, together with associated bioinformatic tools, have enabled large numbers of carbohydrate-acting enzymes to be putatively identified. However, there is a paucity of methods for rapidly screening the biochemical activities of these enzymes, and this is a serious bottleneck in the development of enzyme-reliant bio-refining processes.
We have developed a new generation of multi-coloured chromogenic polysaccharide and protein substrates that can be used in cheap, convenient and high-throughput multiplexed assays. In addition, we have produced substrates of biomass materials in which the complexity of plant cell walls is partially maintained.
We show that these substrates can be used to screen the activities of glycosyl hydrolases, lytic polysaccharide monooxygenases and proteases and provide insight into substrate availability within biomass. We envisage that the assays we have developed will be used primarily for first-level screening of large numbers of putative carbohydrate-acting enzymes, and the assays have the potential to be incorporated into fully or semi-automated robotic enzyme screening systems.
Electronic supplementary material
The online version of this article (doi:10.1186/s13068-015-0250-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4428106  PMID: 25969695
Chromogenic substrates; Carbohydrate-active enzymes; High-throughput screening; Biomass degradation; Plant cell walls; Lytic polysaccharide monooxygenases
19.  Leukocyte telomere length and left ventricular function after acute ST-elevation myocardial infarction: data from the glycometabolic intervention as adjunct to primary coronary intervention in ST elevation myocardial infarction (GIPS-III) trial 
Clinical Research in Cardiology  2015;104(10):812-821.
Telomere length has been associated with coronary artery disease and heart failure. We studied whether leukocyte telomere length is associated with left ventricular ejection fraction (LVEF) after ST-elevation myocardial infarction (STEMI).
Methods and results
Leukocyte telomere length (LTL) was determined using the monochrome multiplex quantitative PCR method in 353 patients participating in the glycometabolic intervention as adjunct to primary percutaneous coronary intervention in STEMI III trial. LVEF was assessed by magnetic resonance imaging. The mean age of patients was 58.9 ± 11.6 years, 75 % were male. In age- and gender-adjusted models, LTL at baseline was significantly associated with age (beta ± standard error; −0.33 ± 0.01; P < 0.01), gender (0.15 ± 0.03; P < 0.01), TIMI flow pre-PCI (0.05 ± 0.03; P < 0.01), TIMI flow post-PCI (0.03 ± 0.04; P < 0.01), myocardial blush grade (−0.05 ± 0.07; P < 0.01), serum glucose levels (−0.11 ± 0.01; P = 0.03), and total leukocyte count (−0.11 ± 0.01; P = 0.04). At 4 months after STEMI, LVEF was well preserved (54.1 ± 8.4 %) and was not associated with baseline LTL (P = 0.95). Baseline LTL was associated with n-terminal pro-brain natriuretic peptide (NT-proBNP) at 4 months (−0.14 ± 0.01; P = 0.02), albeit not independent for age and gender.
Our study does not support a role for LTL as a causal factor related to left ventricular ejection fraction after STEMI.
PMCID: PMC4580719  PMID: 25840550
Telomeres; ST-elevation myocardial infarction; Left ventricular ejection fraction; Metformin
20.  DPP4 inhibition attenuates filtration barrier injury and oxidant stress in the Zucker obese rat 
Obesity (Silver Spring, Md.)  2014;22(10):2172-2179.
Obesity-related glomerulopathy is characterized initially by glomerular hyperfiltration with hypertrophy and then development of proteinuria. Putative mechanisms include endothelial dysfunction and filtration barrier injury due to oxidant stress and immune activation. There has been recent interest in targeting dipeptidyl peptidase 4 (DPP4) enzyme due to increasing role in non-enzymatic cellular processes.
We utilized the Zucker obese (ZO) rat, (aged 8 weeks) fed a normal chow or diet containing the DPP4 inhibitor linagliptin for 8 weeks (83mg/kg rat chow).
Compared to lean controls, there were increases in plasma DPP4 activity along with proteinuria in ZO rats. ZO rats further displayed increases in glomerular size and podocyte foot process effacement. These findings occurred in parallel with decreased endothelial stromal-derived factor-1α (SDF-1α), increased oxidant markers, and tyrosine phosphorylation of nephrin and serine phosphorylation of the mammalian target of rapamycin (mTOR). DPP4 inhibition improved proteinuria along with filtration barrier remodeling, circulating and kidney tissue DPP4 activity, increased active glucagon like peptide–1 (GLP-1) as well as SDF-1α, and improved oxidant markers and the podocyte-specific protein nephrin.
These data support a role for DPP4 in glomerular filtration function and targeting DPP4 with inhibition improves oxidant stress-related glomerulopathy and associated proteinuria.
PMCID: PMC4180797  PMID: 24995775
SDF-1α; linagliptin; NADPH oxidase; DPP4 activity; obesity
21.  Design and Characterization of a Membrane Protein Unfolding Platform in Lipid Bilayers 
PLoS ONE  2015;10(3):e0120253.
Accurate measurement of membrane protein stability—and particularly how it may vary as a result of disease-phenotypic mutations—ideally requires a denaturant that can unfold a membrane-embedded structure while leaving the solubilizing environment unaffected. The steric trap method fulfills this requirement by using monovalent streptavidin (mSA) molecules to unfold membrane proteins engineered with two spatially close biotin tags. Here we adapted this method to an 87-residue helix-loop-helix (hairpin) construct derived from helices 3 and 4 in the transmembrane domain of the human cystic fibrosis transmembrane conductance regulator (CFTR), wherein helix-helix tertiary interactions are anticipated to confer a portion of construct stability. The wild type CFTR TM3/4 hairpin construct was modified with two accessible biotin tags for mSA-induced unfolding, along with two helix-terminal pyrene labels to monitor loss of inter-helical contacts by pyrene excimer fluorescence. A series of eight constructs with biotin tags at varying distances from the helix-terminal pyrene labels were expressed, purified and labeled appropriately; all constructs exhibited largely helical circular dichroism spectra. We found that addition of mSA to an optimized construct in lipid vesicles led to a complete and reversible loss in pyrene excimer fluorescence and mSA binding, and hence hairpin unfolding—results further supported by SDS-PAGE visualization of mSA bound and unbound species. While some dimeric/oligomeric populations persist that may affect quantitation of the unfolding step, our characterization of the design yields a promising prototype of a future platform for the systematic study of membrane protein folding in a lipid bilayer environment.
PMCID: PMC4370600  PMID: 25799099
22.  MDCT evaluation of aortic root and aortic valve prior to TAVI. What is the optimal imaging time point in the cardiac cycle? 
European Radiology  2015;25(7):1975-1983.
To determine the optimal imaging time point for transcatheter aortic valve implantation (TAVI) therapy planning by comprehensive evaluation of the aortic root.
Multidetector-row CT (MDCT) examination with retrospective ECG gating was retrospectively performed in 64 consecutive patients referred for pre-TAVI assessment. Eighteen different parameters of the aortic root were evaluated at 11 different time points in the cardiac cycle. Time points at which maximal (or minimal) sizes were determined, and dimension differences to other time points were evaluated. Theoretical prosthesis sizing based on different measurements was compared.
Largest dimensions were found between 10 and 20 % of the cardiac cycle for annular short diameter (10 %); mean diameter (10 %); effective diameter and circumference-derived diameter (20 %); distance from the annulus to right coronary artery ostium (10 %); aortic root at the left coronary artery level (20 %); aortic root at the widest portion of coronary sinuses (20 %); and right leaflet length (20 %). Prosthesis size selection differed depending on the chosen measurements in 25–75 % of cases.
Significant changes in anatomical structures of the aortic root during the cardiac cycle are crucial for TAVI planning. Imaging in systole is mandatory to obtain maximal dimensions.
Key Points
• Most aortic root structures undergo significant dimensional changes throughout the cardiac cycle.
• The largest dimensions of aortic parameters should be determined to optimize TAVI.
• Circumference-derived diameter showed maximum dimension at 20 % of the cardiac cycle.
PMCID: PMC4457917  PMID: 25708961
Aortic valve; Aortic valve stenosis; Multidetector computed tomography; Cardiac gated imaging technique; Heart valve prosthesis implantation
23.  The pathophysiology of hypertension in patients with obesity 
Nature reviews. Endocrinology  2014;10(6):364-376.
The combination of obesity and hypertension is associated with high morbidity and mortality because it leads to cardiovascular and kidney disease. Potential mechanisms linking obesity to hypertension include dietary factors, metabolic, endothelial and vascular dysfunction, neuroendocrine imbalances, sodium retention, glomerular hyperfiltration, proteinuria, and maladaptive immune and inflammatory responses. Visceral adipose tissue also becomes resistant to insulin and leptin and is the site of altered secretion of molecules and hormones such as adiponectin, leptin, resistin, TNF and IL-6, which exacerbate obesity-associated cardiovascular disease. Accumulating evidence also suggests that the gut microbiome is important for modulating these mechanisms. Uric acid and altered incretin or dipeptidyl peptidase 4 activity further contribute to the development of hypertension in obesity. The pathophysiology of obesity-related hypertension is especially relevant to premenopausal women with obesity and type 2 diabetes mellitus who are at high risk of developing arterial stiffness and endothelial dysfunction. In this Review we discuss the relationship between obesity and hypertension with special emphasis on potential mechanisms and therapeutic targeting that might be used in a clinical setting.
PMCID: PMC4308954  PMID: 24732974
24.  Genomic Features of a Bumble Bee Symbiont Reflect Its Host Environment 
Applied and Environmental Microbiology  2014;80(13):3793-3803.
Here, we report the genome of one gammaproteobacterial member of the gut microbiota, for which we propose the name “Candidatus Schmidhempelia bombi,” that was inadvertently sequenced alongside the genome of its host, the bumble bee, Bombus impatiens. This symbiont is a member of the recently described bacterial order Orbales, which has been collected from the guts of diverse insect species; however, “Ca. Schmidhempelia” has been identified exclusively with bumble bees. Metabolic reconstruction reveals that “Ca. Schmidhempelia” lacks many genes for a functioning NADH dehydrogenase I, all genes for the high-oxygen cytochrome o, and most genes in the tricarboxylic acid (TCA) cycle. “Ca. Schmidhempelia” has retained NADH dehydrogenase II, the low-oxygen specific cytochrome bd, anaerobic nitrate respiration, mixed-acid fermentation pathways, and citrate fermentation, which may be important for survival in low-oxygen or anaerobic environments found in the bee hindgut. Additionally, a type 6 secretion system, a Flp pilus, and many antibiotic/multidrug transporters suggest complex interactions with its host and other gut commensals or pathogens. This genome has signatures of reduction (2.0 megabase pairs) and rearrangement, as previously observed for genomes of host-associated bacteria. A survey of wild and laboratory B. impatiens revealed that “Ca. Schmidhempelia” is present in 90% of individuals and, therefore, may provide benefits to its host.
PMCID: PMC4054214  PMID: 24747890
25.  The impact of coronary artery disease risk loci on ischemic heart failure severity and prognosis: association analysis in the COntrolled ROsuvastatin multiNAtional trial in heart failure (CORONA) 
BMC Medical Genetics  2014;15:140.
Recent genome-wide association studies have identified multiple loci that are associated with an increased risk of developing coronary artery disease (CAD). The impact of these loci on the disease severity and prognosis of ischemic heart failure due to CAD is currently unknown.
We undertook association analysis of 7 single nucleotide polymorphism (rs599839, rs17465637, rs2972147, rs6922269, rs1333049, rs501120, and rs17228212) at 7 well established CAD risk loci (1p13.3, 1q41, 2q36.3, 6q25.1, 9p21.3, 10q11.21, and 15q22.33, respectively) in 3,320 subjects diagnosed with systolic heart failure of ischemic aetiology and participating in the COntrolled ROsuvastatin multiNAtional Trial in Heart Failure (CORONA) trial. The primary outcome was the composite of time to first event of cardiovascular death, non-fatal myocardial infarction and non-fatal stroke, secondary outcomes included mortality and hospitalization due to worsening heart failure.
None of the 7 loci were significantly associated with the primary composite endpoint of the CORONA trial (death from cardiovascular cases, nonfatal myocardial infarction, and nonfatal stroke). However, the 1p13.3 locus (rs599839) showed evidence for association with all-cause mortality (after adjustment for covariates; HR 0.74, 95%CI [0.61 to 0.90]; P = 0.0025) and we confirmed the 1p13.3 locus (rs599839) to be associated with lipid parameters (total cholesterol (P = 1.1x10−4), low-density lipoprotein levels (P = 3.5 × 10−7) and apolipoprotein B (P = 2.2 × 10−10)).
Genetic variants strongly associated with CAD risk are not associated with the severity and outcome of ischemic heart failure. The observed association of the 1p13.3 locus with all-cause mortality requires confirmation in further studies.
Electronic supplementary material
The online version of this article (doi:10.1186/s12881-014-0140-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4412120  PMID: 25528061
Coronary artery disease; Heart failure; Genetics; Healthy ageing; SNP

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