Enhanced Levels of Adiposity, Stretch and Fibrosis Markers in Patients with Coexistent Heart Failure and Atrial Fibrillation.

The coexistence of heart failure (HF) and atrial fibrillation (AF) worsens the prognosis of patients. We aimed to study the inflammation, metabolism, adiposity, and fibrosis markers on epicardial and subcutaneous fat and blood, and their relationship with HF and AF. Samples from 185 patients undergoing cardiac surgery were collected. Levels of multi-markers on fat biopsies and plasma were analyzed. Patients were grouped by HF or AF presence. Plasma adiposity markers were increased in AF patients, while increased stretch markers correlated with HF. Patients with both AF and HF had higher ANP and GDF-15 levels. After excluding AF patients, plasma FABP4 was identified as the main HF predictor. Fat biopsies from AF patients showed an enhanced inflammatory profile. Higher levels of adiposity markers are associated with AF or HF, and higher stretch and fibrosis markers with combined AF and HF, suggesting a role of adiposity-fibrosis pathway in HF and AF coexistence.

Clinical Profiling and Biomarkers for Post-Operative Atrial Fibrillation Prediction in Patients Undergoing Cardiac Surgery.

Post-operative atrial fibrillation (POAF) is the most common arrhythmia in the post-operative period after cardiac surgery. We aim to investigate the main clinical, local, and/or peripheral biochemical and molecular predictors for POAF in patients undergoing coronary and/or valve surgery. Between August 2020 and September 2022, consecutive patients undergoing cardiac surgery without previous history of AF were studied. Clinical variables, plasma, and biological tissues (epicardial and subcutaneous fat) were obtained before surgery. Pre-operative markers associated with inflammation, adiposity, atrial stretch, and fibrosis were analyzed on peripheral and local samples with multiplex assay and real-time PCR. Univariate and multivariate logistic regression analyses were performed in order to identify the main predictors for POAF. Patients were followed-up until hospital discharge. Out of 123 consecutive patients without prior AF, 43 (34.9%) developed POAF during hospitalization. The main predictors were cardiopulmonary bypass time (odds ratio (OR) 1.008 (95% confidence interval (CI), 1.002-1.013), p = 0.005), and plasma pre-operative orosomucoid levels (OR 1.008 (1.206-5.761). After studying differences regarding sex, orosomucoid was the best predictor for POAF in women (OR 2.639 (95% CI, 1.455-4.788), p = 0.027) but not in men. The results support the pre-operative inflammation pathway as a factor involved in the risk of POAF, mainly in women.

Urban Flood Modeling and Risk Assessment with Limited Observation Data: The Beijing Future Science City of China.

The frequency of urban storms has increased, influenced by the climate changing and urbanization, and the process of urban rainfall runoff has also changed, leading to severe urban waterlogging problems. Against this background, the risk of urban waterlogging was analyzed and assessed accurately, using an urban stormwater model as necessary. Most studies have used urban hydrological models to assess flood risk; however, due to limited flow pipeline data, the calibration and the validation of the models are difficult. This study applied the MIKE URBAN model to build a drainage system model in the Beijing Future Science City of China, where the discharge of pipelines was absent. Three methods, of empirical calibration, formula validation, and validation based on field investigation, were used to calibrate and validate the parameters of the model. After the empirical calibration, the relative error range between the simulated value and the measured value was verified by the formula as within 25%. The simulated runoff depth was consistent with a field survey verified by the method of validation based on field investigation, showing the model has good applicability in the study area. Then, the rainfall scenarios of different return periods were designed and simulated. Simulation results showed that, for the 10-year return period, there are overflow pipe sections in northern and southern regions, and the number of overflow pipe sections in the northern region is more than that in the southern region. For the 20-year return period and 50-year return period, the number of overflow pipe sections and nodes in the northern region increased, while for the 100-year return period, the number of overflow nodes both increased. With the increase in the rainfall return period, the pipe network load increased, the points and sections prone to accumulation and waterlogging increased, and the regional waterlogging risk increased. The southern region is prone to waterlogging because the pipeline network density is higher than that in the northern region and the terrain is low-lying. This study provides a reference for the establishment of rainwater drainage models in regions with similar database limitations and provides a technical reference for the calibration and validation of stormwater models that lack rainfall runoff data.

Synergism between obesity and HFpEF on neutrophils phenotype and its regulation by adipose tissue-molecules and SGLT2i dapagliflozin.

The adiposity invokes innate immune activity, coronary microvascular dysfunction and consequently heart failure preserved ejection fraction (HFpEF). Our aim was to study the neutrophils profile on obesity and cardiovascular disease and its regulation by adipose tissue-secretome and dapagliflozin. We have isolated neutrophils from patients undergoing open heart surgery (19 women and 51 men). Its migration activity was performed with culture-transwell, transcriptional studies of proteolytic enzymes, adhesion molecules or receptors were analysed by real-time PCR and proteomics (from 20 patients) analysis by TripleTOF mass spectrometer. Differentiated HL-60 (dHL-60) was used as a preclinical model on microfluidic for endothelial cells attaching assays and genes regulation with epicardial and subcutaneous fat secretomes from patients (3 women and 9 men) or dapagliflozin 1-10 µM treatments. The transcriptional and proteomics studies have determined higher levels of adhesion molecules in neutrophils from patients with obesity. The adhesion molecule CD11b levels were higher in those patients with the combined obesity and HFpEF factors (1.70 ± 0.06 a.u. without obesity, 1.72 ± 0.04 a.u. obesity or HFpEF without obesity and 1.79 ± 0.08 a.u. obesity and HFpEF; p < .01). While fat-secretome induces its upregulation, dapagliflozin can modulated it. Because CD11b upregulation is associated with higher neutrophils migration and adhesion into endothelial cells, dapagliflozin might modulate this mechanism on patients with obesity and HFpEF.

The Effect of Mineralocorticoid Receptor 3 Antagonists on Anti-Inflammatory and Anti-Fatty Acid Transport Profile in Patients with Heart Failure.

Epicardial fat thickness is associated with cardiovascular disease. Mineralocorticoid receptor antagonist (MRA), a pharmaceutical treatment for CVD, was found to have an effect on adipose tissue. Our aim was to analyse the main epicardial fat genesis and inflammation-involved cell markers and their regulation by risk factors and MRA. We included blood and epicardial or subcutaneous fat (EAT or SAT) from 71 patients undergoing heart surgery and blood from 66 patients with heart failure. Cell types (transcripts or proteins) were analysed by real-time polymerase chain reaction or immunohistochemistry. Plasma proteins were analysed by Luminex technology or enzyme-linked immunoassay. Our results showed an upregulation of fatty acid transporter levels after aldosterone-induced genesis. The MRA intake was the main factor associated with lower levels in epicardial fat. On the contrary, MRA upregulated the levels and its secretion of the anti-inflammatory marker intelectin 1 and reduced the proliferation of epicardial fibroblasts. Our results have shown the local MRA intake effect on fatty acid transporters and anti-inflammatory marker levels and the proliferation rate on epicardial fat fibroblasts. They suggest the role of MRA on epicardial fat genesis and remodelling in patients with cardiovascular disease. Translational perspective: the knowledge of epicardial fat genesis and its modulation by drugs might be useful for improving the treatments of cardiovascular disease.

Bacillus thuringiensis chimeric proteins Cry1A.2 and Cry1B.2 to control soybean lepidopteran pests: New domain combinations enhance insecticidal spectrum of activity and novel receptor contributions.

Two new chimeric Bacillus thuringiensis (Bt) proteins, Cry1A.2 and Cry1B.2, were constructed using specific domains, which provide insecticidal activity against key lepidopteran soybean pests while minimizing receptor overlaps between themselves, current, and soon to be commercialized plant incorporated protectants (PIP's) in soybean. Results from insect diet bioassays demonstrate that the recombinant Cry1A.2 and Cry1B.2 are toxic to soybean looper (SBL) Chrysodeixis includens Walker, velvetbean caterpillar (VBC) Anticarsia gemmatalis Hubner, southern armyworm (SAW) Spodoptera eridania, and black armyworm (BLAW) Spodoptera cosmioides with LC50 values < 3,448 ng/cm2. Cry1B.2 is of moderate activity with significant mortality and stunting at > 3,448 ng/cm2, while Cry1A.2 lacks toxicity against old-world bollworm (OWB) Helicoverpa armigera. Results from disabled insecticidal protein (DIP) bioassays suggest that receptor utilization of Cry1A.2 and Cry1B.2 proteins are distinct from each other and from current, and yet to be commercially available, Bt proteins in soy such as Cry1Ac, Cry1A.105, Cry1F.842, Cry2Ab2 and Vip3A. However, as Cry1A.2 contains a domain common to at least one commercial soybean Bt protein, resistance to this common domain in a current commercial soybean Bt protein could possibly confer at least partial cross resistance to Cry1A2. Therefore, Cry1A.2 and Cry1B.2 should provide two new tools for controlling many of the major soybean insect pests described above.

Bacillus thuringiensis Cry1Da_7 and Cry1B.868 Protein Interactions with Novel Receptors Allow Control of Resistant Fall Armyworms, Spodoptera frugiperda (J.E. Smith).

Two new modified Bacillus thuringiensis (Bt) proteins, Cry1Da_7 and Cry1B.868, with activity against fall armyworms (FAW), Spodoptera frugiperda (J.E. Smith), were evaluated for their potential to bind new insect receptors compared to proteins currently deployed as plant-incorporated protectants (PIPs) in row crops. Results from resistant insect bioassays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that receptor utilizations of the newly modified Cry1Da_7 and Cry1B.868 proteins are distinct from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Accordingly, these two proteins target different insect proteins in FAW midgut cells and when pyramided together should provide durability in the field against this economically important pest.IMPORTANCE There is increased concern with the development of resistance to insecticidal proteins currently expressed in crop plants, especially against high-resistance-risk pests such as fall armyworm (FAW), Spodoptera frugiperda, a maize pest that already has developed resistance to Bacillus thuringiensis (Bt) proteins such as Cry1F. Lepidopteran-specific proteins that bind new insect receptors will be critical in managing current Cry1F-resistant FAW and delaying future resistance development. Results from resistant insect assays, disabled insecticidal protein (DIP) bioassays, and cell-based assays using insect cells expressing individual receptors demonstrate that target receptors of the Cry1Da_7 and Cry1B.868 proteins are different from each other and from those of commercially available Bt proteins such as Cry1F, Cry1A.105, Cry2Ab, and Vip3A. Therefore, pyramiding these two new proteins in maize will provide durable control of this economically important pest in production agriculture.

Development of enzymes for robust aryloxyphenoxypropionate and synthetic auxin herbicide tolerance traits in maize and soybean crops.

BACKGROUND: Effective management of weedy species in agricultural fields is essential for maintaining favorable growing conditions and crop yields. The introduction of genetically modified crops containing herbicide tolerance traits has been a successful additional tool available to farmers to better control weeds. However, weed resistance challenges present a need for additional herbicide tolerance trait options. RESULTS: To help meet this challenge, a new trait that provides tolerance to an aryloxyphenoxypropionate (FOP) herbicide and members of the synthetic auxin herbicide family, such as 2,4-dichlorophenoxyacetic acid (2,4-D), was developed. Development of this herbicide tolerance trait employed an enzyme engineered with robust and specific enzymatic activity for these two herbicide families. This engineering effort utilized a microbial-sourced dioxygenase scaffold to generate variants with improved enzymatic parameters. Additional optimization to enhance in-plant stability of the enzyme enabled an efficacious trait that can withstand the higher temperature conditions often found in field environments. CONCLUSION: Optimized herbicide tolerance enzyme variants with enhanced enzymatic and temperature stability parameters enabled robust herbicide tolerance for two herbicide families in transgenic maize and soybeans. This herbicide tolerance trait for FOP and synthetic auxin herbicides such as 2,4-D could be useful in weed management systems, providing additional tools for farmers to control weeds. © 2019 Society of Chemical Industry.

Disabled insecticidal proteins: A novel tool to understand differences in insect receptor utilization.

The development of insect resistance to pesticides via natural selection is an acknowledged agricultural issue. Likewise, resistance development in target insect populations is a significant challenge to the durability of crop traits conferring insect protection and has driven the need for novel insecticidal proteins (IPs) with alternative mechanism of action (MOA) mediated by different insect receptors. The combination or "stacking" of transgenes encoding different insecticidal proteins in a single crop plant can greatly delay the development of insect resistance, but requires sufficient knowledge of MOA to identify proteins with different receptor preferences. Accordingly, a rapid technique for differentiating the receptor binding preferences of insecticidal proteins is a critical need. This article introduces the Disabled Insecticidal Protein (DIP) method as applied to the well-known family of three-domain insecticidal proteins from Bacillus thuringiensis and related bacteria. These DIP's contain amino acid substitutions in domain 1 that render the proteins non-toxic but still capable of competing with active proteins in insect feeding assays, resulting in a suppression of the expected insecticidal activity. A set of insecticidal proteins with known differences in receptor binding (Cry1Ab3, Cry1Ac.107, Cry2Ab2, Cry1Ca, Cry1A.105, and Cry1A.1088) has been studied using the DIP method, yielding results that are consistent with previous MOA studies. When a native IP and an excess of DIP are co-administered to insects in a feeding assay, the outcome depends on the overlap between their MOAs: if receptors are shared, then the DIP saturates the receptors to which the native protein would ordinarily bind, and acts as an antidote whereas, if there is no shared receptor, the toxicity of the native insecticidal protein is not inhibited. These results suggest that the DIP methodology, employing standard insect feeding assays, is a robust and effective method for rapid MOA differentiation among insecticidal proteins.

Involvement of the N-terminal B-box domain of Arabidopsis BBX32 protein in interaction with soybean BBX62 protein.

Previous studies have demonstrated that Arabidopsis thaliana BBX32 (AtBBX32) represses light signaling in A. thaliana and that expression of AtBBX32 in soybean increases grain yield in multiple locations and multiyear field trials. The BBX32 protein is a member of the B-box zinc finger family from A. thaliana and contains a single conserved Zn(2+)-binding B-box domain at the N terminus. Although the B-box domain is predicted to be involved in protein-protein interactions, the mechanism of interaction is poorly understood. Here, we provide in vitro and in vivo evidence demonstrating the physical and functional interactions of AtBBX32 with another B-box protein, soybean BBX62 (GmBBX62). Deletion analysis and characterization of the purified B-box domain indicate that the N-terminal B-box region of AtBBX32 interacts with GmBBX62. Computational modeling and site-directed mutagenesis of the AtBBX32 B-box region identified specific residues as critical for mediating the interaction between AtBBX32 and GmBBX62. This study defines the plant B-box as a protein interaction domain and offers novel insight into its role in mediating specific protein-protein interactions between different plant B-box proteins.

X-ray vs. NMR structures as templates for computational protein design.

Certain protein-design calculations involve using an experimentally determined high-resolution structure as a template to identify new sequences that can adopt the same fold. This approach has led to the successful design of many novel, well-folded, native-like proteins. Although any atomic-resolution structure can serve as a template in such calculations, most successful designs have used high-resolution crystal structures. Because there are many proteins for which crystal structures are not available, it is of interest whether nuclear magnetic resonance (NMR) templates are also appropriate. We have analyzed differences between using X-ray and NMR templates in side-chain repacking and design calculations. We assembled a database of 29 proteins for which both a high-resolution X-ray structure and an ensemble of NMR structures are available. Using these pairs, we compared the rotamericity, chi(1)-angle recovery, and native-sequence recovery of X-ray and NMR templates. We carried out design using RosettaDesign on both types of templates, and compared the energies and packing qualities of the resulting structures. Overall, the X-ray structures were better templates for use with Rosetta. However, for approximately 20% of proteins, a member of the reported NMR ensemble gave rise to designs with similar properties. Re-evaluating RosettaDesign structures with other energy functions indicated much smaller differences between the two types of templates. Ultimately, experiments are required to confirm the utility of particular X-ray and NMR templates. But our data suggest that the lack of a high-resolution X-ray structure should not preclude attempts at computational design if an NMR ensemble is available.

Modeling backbone flexibility to achieve sequence diversity: the design of novel alpha-helical ligands for Bcl-xL.

Computational protein design can be used to select sequences that are compatible with a fixed-backbone template. This strategy has been used in numerous instances to engineer novel proteins. However, the fixed-backbone assumption severely restricts the sequence space that is accessible via design. For challenging problems, such as the design of functional proteins, this may not be acceptable. Here, we present a method for introducing backbone flexibility into protein design calculations and apply it to the design of diverse helical BH3 ligands that bind to the anti-apoptotic protein Bcl-xL, a member of the Bcl-2 protein family. We demonstrate how normal mode analysis can be used to sample different BH3 backbones, and show that this leads to a larger and more diverse set of low-energy solutions than can be achieved using a native high-resolution Bcl-xL complex crystal structure as a template. We tested several of the designed solutions experimentally and found that this approach worked well when normal mode calculations were used to deform a native BH3 helix structure, but less well when they were used to deform an idealized helix. A subsequent round of design and testing identified a likely source of the problem as inadequate sampling of the helix pitch. In all, we tested 17 designed BH3 peptide sequences, including several point mutants. Of these, eight bound well to Bcl-xL and four others showed weak but detectable binding. The successful designs showed a diversity of sequences that would have been difficult or impossible to achieve using only a fixed backbone. Thus, introducing backbone flexibility via normal mode analysis effectively broadened the set of sequences identified by computational design, and provided insight into positions important for binding Bcl-xL.

Probabilistic approach to the design of symmetric protein quaternary structures.

Probabilistic methods have been developed that estimate the site-specific probabilities of the amino acids in sequences likely to fold to a particular target structure, and such information can be used to guide the de novo design of proteins and to probe sequence variability. An extension of these methods for the design of symmetric homo-oligomeric quaternary structures is presented. The theory is in excellent agreement with the results of studies on exactly solvable lattice models. Application to an atomically detailed representation of proteins verifies the utility of a symmetry assumption, which greatly simplifies and accelerates the calculations. The method may be applied to a wide variety of symmetric and periodic protein structures.