Experimental design principles were applied on cell-free protein synthesis to optimize performance with regard to the expression yield and the incorporation efficiency of amino acid precursors. A versatile screening platform based on batch-mode cell-free expression and central composite design was used. The performance of different extracts (S12 and S30), the concentration dependence of key components and the effect of different additives were investigated. We find that the initial expression yield can be enhanced twofold to threefold in this manner. The improved conditions comprise a modified S12 extract, optimized concentrations of creatine phosphate and key amino acids, as well as introduction of ketoacid additives. Our results show that current cell-free expression technology is far from optimal and that higher yields and increased utilization of the provided precursors are attainable with further optimization.

Tubulointerstitial fibrosis is a major feature of chronic kidney disease. Unilateral ureteral obstruction (UUO) in rodents leads to the development of renal tubulointerstitial fibrosis consistent with histopathological changes observed in advanced chronic kidney disease in humans. The purpose of this study was to assess the effect of inhibiting angiotensin II receptors or Ras activation on early renal fibrotic changes induced by UUO. Animals either received angiotensin II or underwent UUO. UUO animals received either losartan, atorvastatin, and farnesyl transferase inhibitor (FTI) L-744,832, or chaetomellic acid A (ChA). Levels of activated Ras, phospho-ERK1/2, phospho-Akt, fibronectin, and α-smooth muscle actin were subsequently quantified in renal tissue by ELISA, Western blot, and/or immunohistochemistry. Our results demonstrate that administration of angiotensin II induces activation of the small GTPase Ras/Erk/Akt signaling system, suggesting an involvement of angiotensin II in the early obstruction-induced activation of renal Ras. Furthermore, upstream inhibition of Ras signalling by blocking either angiotensin AT1 type receptor or by inhibiting Ras prenylation (atorvastatin, FTI o ChA) reduced the activation of the Ras/Erk/Akt signaling system and decreased the early fibrotic response in the obstructed kidney. This study points out that pharmacological inhibition of Ras activation may hold promise as a future strategy in the prevention of renal fibrosis.

The epithelial layer within the colon represents a physical barrier between the luminal contents and its underlying mucosa. It plays a pivotal role in mucosal homeostasis, and both tolerance and anti-pathogenic immune responses. Identifying signals of inflammation initiation and responses to stimuli from within the epithelial layer is critical to understanding the molecular pathways underlying disease pathology. This study validated a method to isolate and analyze epithelial populations, enabling investigations of epithelial function and response in a variety of disease setting.

Roux-en-Y gastric bypass (LRYGB) has weight-independent effects on glycemia in obese type 2 diabetic patients, whereas sleeve gastrectomy (LSG) is less well characterized. This study aims to compare early weight-independent and later weight-dependent glycemic effects of LRYGB and LSG.

Gastric bypass surgery is a very effective treatment of obesity and type 2 diabetes. However, very few eligible patients are offered surgery. Some patients also prefer less invasive approaches. We aimed to study the effects of the Sleeveballoon - a new device combining an intragastric balloon with a connecting sleeve, which covers the duodenal and proximal jejunal mucosa - on insulin sensitivity, glycemic control, body weight and body fat distribution.

Multiple studies demonstrate an albuminuria-lowering impact of Roux-en-Y gastric bypass surgery, but neither evaluation of its penetrance across different baseline levels of albuminuria nor its association with alterations in podocyte phenotype has previously been reported.

Intra-abdominal or visceral obesity is associated with insulin resistance and an increased risk for cardiovascular disease. This study aimed to compare the effects of semaglutide 1.0 mg and canagliflozin 300 mg on body composition in a subset of participants from the SUSTAIN 8 Phase IIIB, randomised double-blind trial who underwent whole-body dual-energy x-ray absorptiometry (DXA) scanning.

Despite substantial research efforts, the mechanisms proposed to explain weight loss after gastric bypass (RYGB) and sleeve gastrectomy (SL) do not explain the large individual variation seen after these treatments. A complex set of factors are involved in the onset and development of obesity and these may also be relevant for the understanding of why success with treatments vary considerably between individuals. This calls for explanatory models that take into account not only biological determinants but also behavioral, affective and contextual factors. In this prospective study, we recruited 47 women and 8 men, aged 25-56 years old, with a BMI of 45.8 ± 7.1 kg/m2 from the waiting list for RYGB and SL at Køge hospital, Denmark. Pre-surgery and 1.5, 6 and 18 months after surgery we assessed various endpoints spanning multiple domains. Endpoints were selected on basis of previous studies and include: physiological measures: anthropometrics, vital signs, biochemical measures and appetite hormones, genetics, gut microbiota, appetite sensation, food and taste preferences, neural sensitivity, sensory perception and movement behaviors; psychological measures: general psychiatric symptom-load, depression, eating disorders, ADHD, personality disorder, impulsivity, emotion regulation, attachment pattern, general self-efficacy, alexithymia, internalization of weight bias, addiction, quality of life and trauma; and sociological and anthropological measures: sociodemographic measures, eating behavior, weight control practices and psycho-social factors.Joining these many endpoints and methodologies from different scientific disciplines and creating a multi-dimensional predictive model has not previously been attempted. Data on the primary endpoint are expected to be published in 2018.

No abstract available

To establish whether obesity involves activation of endogenous ciliary neurotrophic factor (CNTF) signalling, we evaluated its plasma levels in patients with obesity and correlated its values with the major clinical and haematological indices of obesity, insulin resistance and systemic inflammation. This study involved 118 subjects: 39 healthy controls (19 men), 39 subjects with obesity (19 men) and 40 subjects with obesity and diabetes (20 men). Plasma CNTF and CNTF receptor α (CNTFRα) were measured using commercial ELISA kits. The results showed that plasma CNTF was significantly higher in males and females with obesity with and without diabetes than in healthy subjects. Women consistently exhibited higher levels of circulating CNTF. In both genders, CNTF levels correlated significantly and positively with obesity (BMI, WHR, leptin), diabetes (fasting insulin, HOMA index and HbA1c) and inflammation (IL-6 and hsCRP) indices. Circulating CNTFRα and the CNTF/CNTFRα molar ratio tended to be higher in the patient groups than in controls. In conclusion, endogenous CNTF signalling is activated in human obesity and may help counteract some adverse effects of obesity. Studies involving a higher number of selected patients may reveal circulating CNTF and/or CNTFRα as potential novel diagnostic and/or prognostic markers of obesity, diabetes and associated diseases.

Rationale: Galectin-3 (Gal-3) drives fibrosis during chronic lung injury, however, its role in acute lung injury (ALI) remains unknown. Effective pharmacological therapies available for ALI are limited; identifying novel concepts in treatment is essential. GB0139 is a Gal-3 inhibitor currently under clinical investigation for the treatment of idiopathic pulmonary fibrosis. We investigate the role of Gal-3 in ALI and evaluate whether its inhibition with GB0139 offers a protective role. The effect of GB0139 on ALI was explored in vivo and in vitro. Methods: The pharmacokinetic profile of intra-tracheal (i.t.) GB0139 was investigated in C57BL/6 mice to support the daily dosing regimen. GB0139 (1-30 µg) was then assessed following acute i.t. lipopolysaccharide (LPS) and bleomycin administration. Histology, broncho-alveolar lavage fluid (BALf) analysis, and flow cytometric analysis of lung digests and BALf were performed. The impact of GB0139 on cell activation and apoptosis was determined in vitro using neutrophils and THP-1, A549 and Jurkat E6 cell lines. Results: GB0139 decreased inflammation severity via a reduction in neutrophil and macrophage recruitment and neutrophil activation. GB0139 reduced LPS-mediated increases in interleukin (IL)-6, tumor necrosis factor alpha (TNFα) and macrophage inflammatory protein-1-alpha. In vitro, GB0139 inhibited Gal-3-induced neutrophil activation, monocyte IL-8 secretion, T cell apoptosis and the upregulation of pro-inflammatory genes encoding for IL-8, TNFα, IL-6 in alveolar epithelial cells in response to mechanical stretch. Conclusion: These data indicate that Gal-3 adopts a pro-inflammatory role following the early stages of lung injury and supports the development of GB0139, as a potential treatment approach in ALI.

Galectin-3 (Gal-3) is a β-galactoside-binding lectin that is highly expressed within the tumor microenvironment of aggressive cancers and has been suggested to predict a poor response to immune checkpoint therapy with the anti-PD-1 monoclonal antibody pembrolizumab. We aimed to assess if the effect of Gal-3 was a result of direct interaction with the immune checkpoint receptor.

The anti-apoptotic B cell CLL/lymphoma-2 (Bcl-2) protein is a key player in the regulation of programmed cell death and is linked to various types of cancer and their resistance to drug treatment. Biophysical and structural studies of the full-length intact Bcl-2 have been hampered due to difficulties in expression and severe solubility problems, precluding isolation of this hydrophobic membrane protein. Therefore, previous work has so far mainly been carried out using structurally modified Bcl-2 variants, lacking the transmembrane region. Thus, biophysical information regarding the full-length protein is still missing. Here, a protocol is presented for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)). A batch-based cell-free expression system, using extract isolated from Escherichia coli (E. coli) was employed to produce recombinant protein encoded by an optimized gene sequence. Presence of polyoxyethylene-(20)-cetyl-ether (Brij-58) in the reaction mixture and subsequently in the immobilized metal-affinity purification steps was crucial to keep Bcl-2(2) soluble. The obtained yield was 0.25-0.3mg per ml of cell-free reaction. Far-UV circular dichroism (CD) spectroscopy confirmed the α-helical structure of the purified protein, characteristic for members of the Bcl-2 protein family.

We investigate the feasibility of performing functional MRI (fMRI) at ultralow field (ULF) with a Superconducting QUantum Interference Device (SQUID), as used for detecting magnetoencephalography (MEG) signals from the human head. While there is negligible magnetic susceptibility variation to produce blood oxygenation level-dependent (BOLD) contrast at ULF, changes in cerebral blood volume (CBV) may be a sensitive mechanism for fMRI given the five-fold spread in spin-lattice relaxation time (T1) values across the constituents of the human brain. We undertook simulations of functional signal strength for a simplified brain model involving activation of a primary cortical region in a manner consistent with a blocked task experiment. Our simulations involve measured values of T1 at ULF and experimental parameters for the performance of an upgraded ULFMRI scanner. Under ideal experimental conditions we predict a functional signal-to-noise ratio of between 3.1 and 7.1 for an imaging time of 30 min, or between 1.5 and 3.5 for a blocked task experiment lasting 7.5 min. Our simulations suggest it may be feasible to perform fMRI using a ULFMRI system designed to perform MRI and MEG in situ.

Biobanks are important infrastructures for life science research. Optimal sample handling regarding e.g. collection and processing of biological samples is highly complex, with many variables that could alter sample integrity and even more complex when considering multiple study centers or using legacy samples with limited documentation on sample management. Novel means to understand and take into account such variability would enable high-quality research on archived samples.

To determine whether rats reaching the same body mass, having been fed either a low-fat (LFD) or a high-fat diet (HFD), differ in white adipose tissue (WAT) deposition.

Depending on their protein content, single meals can rapidly influence the uptake of amino acids into the brain and thereby modify brain functions. The current study investigates the effects of two different amino acids on the human gut-brain system, using a multimodal approach, integrating physiological and neuroimaging data. In a randomized, placebo-controlled trial, L-tryptophan, L-leucine, glucose and water were administered directly into the gut of 20 healthy subjects. Functional MRI (fMRI) in a resting state paradigm (RS), combined with the assessment of insulin and glucose blood concentration, was performed before and after treatment. Independent component analysis with dual regression technique was applied to RS-fMRI data. Results were corrected for multiple comparisons. In comparison to glucose and water, L-tryptophan consistently modifies the connectivity of the cingulate cortex in the default mode network, of the insula in the saliency network and of the sensory cortex in the somatosensory network. L-leucine has lesser effects on these functional networks. L-tryptophan and L-leucine also modified plasma insulin concentration. Finally, significant correlations were found between brain modifications after L-tryptophan administration and insulin plasma levels. This study shows that acute L-tryptophan and L-leucine intake directly influence the brain networks underpinning the food-reward system and appetite regulation.

In the Microvascular Outcomes after Metabolic Surgery randomised clinical trial (MOMS RCT, NCT01821508), combined metabolic surgery (gastric bypass) plus medical therapy (CSM) was superior to medical therapy alone (MTA) as a means of achieving albuminuria remission at 2-year follow-up in patients with obesity and early diabetic kidney disease (DKD). In the present study, we assessed the urinary 1H-NMR metabolome in a subgroup of patients from both arms of the MOMS RCT at baseline and 6-month follow-up. Whilst CSM and MTA both reduced the urinary excretion of sugars, CSM generated a distinctive urinary metabolomic profile characterised by increases in host-microbial co-metabolites (N-phenylacetylglycine, trimethylamine N-oxide, and 4-aminobutyrate (GABA)) and amino acids (arginine and glutamine). Furthermore, reductions in aromatic amino acids (phenylalanine and tyrosine), as well as branched-chain amino acids (BCAAs) and related catabolites (valine, leucine, 3-hydroxyisobutyrate, 3-hydroxyisovalerate, and 3-methyl-2-oxovalerate), were observed following CSM but not MTA. Improvements in BMI did not correlate with improvements in metabolic and renal indices following CSM. Conversely, urinary metabolites changed by CSM at 6 months were moderately to strongly correlated with improvements in blood pressure, glycaemia, triglycerides, and albuminuria up to 24 months following treatment initiation, highlighting the potential involvement of these shifts in the urinary metabolomic profile in the metabolic and renoprotective effects of CSM.

Food intake normally stimulates release of satiety and insulin-stimulating intestinal hormones, such as glucagon-like peptide (GLP)-1. This response is blunted in obese insulin resistant subjects, but is rapidly restored following Roux-en-Y gastric bypass (RYGB) surgery. We hypothesised this to be a result of the metabolic changes taking place in the small intestinal mucosa following the anatomical rearrangement after RYGB surgery, and aimed at identifying such mechanisms.

Galectin-3 is a carbohydrate-binding protein central to regulating mechanisms of diseases such as fibrosis, cancer, metabolic, inflammatory, and heart disease. We recently found a high affinity (nM) thiodigalactoside GB0139 which currently is in clinical development (PhIIb) as an inhaled treatment of idiopathic pulmonary fibrosis. To enable treatment of systemically galectin-3 driven disease, we here present the first series of selective galectin-3 inhibitors combining high affinity (nM) with oral bioavailability. This was achieved by optimizing galectin-3 specificity and physical chemical parameters for a series of disubstituted monogalactosides. Further characterization showed that this class of compounds reduced profibrotic gene expression in liver myofibroblasts and displayed antifibrotic activity in CCl4-induced liver fibrosis and bleomycin-induced lung fibrosis mouse models. On the basis of the overall pharmacokinetic, pharmacodynamic, and safety profile, GB1211 was selected as the clinical candidate and is currently in phase IIa clinical trials as a potential therapy for liver cirrhosis and cancer.