Semi-rational engineering of Adh2 for improved methanol utilization in Komagataella phaffii

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Engineering A-type Dye-Decolorizing Peroxidases by Modification of a Conserved Glutamate Residue

Funding Information: . Financial support by the Austrian Science Fund (FWF) through the doctoral program Biomolecular Technology of Proteins (BioToP, grant number W1224), the Austrian Federal Ministry for Digital and Economic Affairs, the National Foundation for Research, Technology and Development, the Christian Doppler Research Association, Funda\u00E7\u00E3o para a Ci\u00EAncia e Tecnologia (FCT), Portugal, grants FCT 2022.02027.PTDC, MOSTMICRO\u2010ITQB (UIDB/04612/2020 and UIDP/04612/2020), LS4FUTURE (LA/P/0087/2020) are gratefully acknowledged Publisher Copyright: © 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.Dye-decolorizing peroxidases (DyPs) are recently identified microbial enzymes that have been used in several Biotechnology applications from wastewater treatment to lignin valorization. However, their properties and mechanism of action still have many open questions. Their heme-containing active site is buried by three conserved flexible loops with a putative role in modulating substrate access and enzyme catalysis. Here, we investigated the role of a conserved glutamate residue in stabilizing interactions in loop 2 of A-type DyPs. First, we did site saturation mutagenesis of this residue, replacing it with all possible amino acids in bacterial DyPs from Bacillus subtilis (BsDyP) and from Kitasatospora aureofaciens (KaDyP1), the latter being characterized here for the first time. We screened the resulting libraries of variants for activity towards ABTS and identified variants with increased catalytic efficiency. The selected variants were purified and characterized for activity and stability. We furthermore used Molecular Dynamics simulations to rationalize the increased catalytic efficiency and found that the main reason is the electron channeling becoming easier from surface-exposed tryptophans. Based on our findings, we also propose that this glutamate could work as a pH switch in the wild-type enzyme, preventing intracellular damage.publishersversionpublishe

Popliteal Cysts in Paediatric Patients: Clinical Characteristics and Imaging Features on Ultrasound and MRI

Popliteal cysts, or Baker cysts, are considered rare in children and may exhibit particular features, as compared with adults. We studied data from 80 paediatric patients with 55 Baker cysts, examined over a period of 7 years, and correlated clinical presentation with findings on ultrasonography and MRI. Prevalence of popliteal cysts was 57% in arthritic knees, 58% with hypermobility syndrome, and 28% without risk factors. Only one patient had a trauma history and showed an ipsilateral cyst. Mean cyst volume was 3.4 mL; cysts were larger in boys. Patients with arthritis had echogenic cysts in 53%. Cyst communication with the joint space was seen in 64% on ultrasonography and 86% on MRI. In conclusion, Baker cysts are a common finding in a clinically preselected paediatric population. Children with Baker cysts should be assessed for underlying arthritis and inherited joint hypermobility, while sporadic Baker cysts appear to be common, as well

Comprehensive Computational Model for Coupled Fluid Flow, Mass Transfer, and Light Supply in Tubular Photobioreactors Equipped with Glass Sponges

The design and optimization of photobioreactor(s) (PBR) benefit from the development of robust and quantitatively accurate computational fluid dynamics (CFD) models, which incorporate the complex interplay of fundamental phenomena. In the present work, we propose a comprehensive computational model for tubular photobioreactors equipped with glass sponges. The simulation model requires a minimum of at least three submodels for hydrodynamics, light supply, and biomass kinetics, respectively. First, by modeling the hydrodynamics, the light–dark cycles can be detected and the mixing characteristics of the flow (besides the mass transport) can be analyzed. Second, the radiative transport model is deployed to predict the local light intensities according to the wavelength of the light and scattering characteristics of the culture. The third submodel implements the biomass growth kinetic by coupling the local light intensities to hydrodynamic information of the CO2 concentration, which allows to predict the algal growth. In combination, the novel mesoscopic simulation model is applied to a tubular PBR with transparent walls and an internal sponge structure. We showcase the coupled simulation results and validate specific submodel outcomes by comparing the experiments. The overall flow velocity, light distribution, and light intensities for individual algae trajectories are extracted and discussed. Conclusively, such insights into complex hydrodynamics and homogeneous illumination are very promising for CFD-based optimization of PBR

Comprehensive Computational Model for Coupled Fluid Flow, Mass Transfer, and Light Supply in Tubular Photobioreactors Equipped with Glass Sponges

The design and optimization of photobioreactor(s) (PBR) benefit from the development of robust and quantitatively accurate computational fluid dynamics (CFD) models, which incorporate the complex interplay of fundamental phenomena. In the present work, we propose a comprehensive computational model for tubular photobioreactors equipped with glass sponges. The simulation model requires a minimum of at least three submodels for hydrodynamics, light supply, and biomass kinetics, respectively. First, by modeling the hydrodynamics, the light–dark cycles can be detected and the mixing characteristics of the flow (besides the mass transport) can be analyzed. Second, the radiative transport model is deployed to predict the local light intensities according to the wavelength of the light and scattering characteristics of the culture. The third submodel implements the biomass growth kinetic by coupling the local light intensities to hydrodynamic information of the CO2 concentration, which allows to predict the algal growth. In combination, the novel mesoscopic simulation model is applied to a tubular PBR with transparent walls and an internal sponge structure. We showcase the coupled simulation results and validate specific submodel outcomes by comparing the experiments. The overall flow velocity, light distribution, and light intensities for individual algae trajectories are extracted and discussed. Conclusively, such insights into complex hydrodynamics and homogeneous illumination are very promising for CFD-based optimization of PBR

Characterization of Von Willebrand Factor Multimer Structure in Patients With Severe Aortic Stenosis

Acquired von Willebrand syndrome (AVWS) associated with severe aortic stenosis (AS) has been frequently subclassified into a subtype 2A based on the deficiency of high-molecular-weight (HMW) multimers as it is seen in inherited von Willebrand disease (VWD) type 2A. However, the multimeric phenotype of VWD type 2A does not only include an HMW deficiency but also a decrease in intermediate-molecular-weight (IMW) multimers and an abnormal inner triplet band pattern. These additional characteristics have not been evaluated in AVWS associated with severe AS. Therefore, we recruited N = 31 consecutive patients with severe AS and performed a high-resolution Western blot with densitometrical band quantification to characterize the von Willebrand factor (VWF) multimeric structure and reevaluate the AVWS subtype classification. Study patients showed an isolated HMW VWF multimer deficiency without additional abnormalities of the IMW portions and the inner triplet structure in 65%. In conclusion, the multimeric pattern of AVWS associated with severe AS does neither resemble that seen in AVWS type 2A nor that seen in inherited VWD type 2A. Therefore, a subclassification into a type 2A should not be used

TAPBPR mediates peptide dissociation from MHC class I using a leucine lever.

Tapasin and TAPBPR are known to perform peptide editing on major histocompatibility complex class I (MHC I) molecules; however, the precise molecular mechanism(s) involved in this process remain largely enigmatic. Here, using immunopeptidomics in combination with novel cell-based assays that assess TAPBPR-mediated peptide exchange, we reveal a critical role for the K22-D35 loop of TAPBPR in mediating peptide exchange on MHC I. We identify a specific leucine within this loop that enables TAPBPR to facilitate peptide dissociation from MHC I. Moreover, we delineate the molecular features of the MHC I F pocket required for TAPBPR to promote peptide dissociation in a loop-dependent manner. These data reveal that chaperone-mediated peptide editing on MHC I can occur by different mechanisms dependent on the C-terminal residue that the MHC I accommodates in its F pocket and provide novel insights that may inform the therapeutic potential of TAPBPR manipulation to increase tumour immunogenicity.Wellcome PhD studentship (109076/Z/15/A). Wellcome Senior Research Fellowship (104647/Z/14/Z) South African Medical Research Council Bosch-Forschungsstiftung. Royal Society University Research Fellowship (UF100371)

The synthetic Tie2 agonist peptide vasculotide protects against vascular leakage and reduces mortality in murine abdominal sepsis

Introduction: Angiopoietin-1 (Angpt1), the natural agonist ligand for the endothelial Tie2 receptor, is a non-redundant endothelial survival and vascular stabilization factor that reduces endothelial permeability and inhibits leukocyte-endothelium interactions. Here we evaluate the efficacy of a novel polyethylene glycol (PEG)-clustered Tie2 agonist peptide, vasculotide (VT), to protect against vascular leakage and mortality in a murine model of polymicrobial abdominal sepsis. Methods: Polymicrobial abdominal sepsis in C57BL6 mice was induced by cecal-ligation-and-puncture (CLP). Mice were treated with different dosages of VT or equal volume of phosphate-buffered saline (PBS). Sham-operated animals served as time-matched controls. Results: Systemic administration of VT induced long-lasting Tie2 activation in vivo. VT protected against sepsis-induced endothelial barrier dysfunction, as evidenced by attenuation of vascular leakage and leukocyte transmigration into the peritoneal cavity. Histological analysis revealed that VT treatment ameliorated leukocyte infiltration in kidneys of septic mice, probably due to reduced endothelial adhesion molecule expression. VT-driven effects were associated with significantly improved organ function and reduced circulating cytokine levels. The endothelial-specific action of VT was supported by additional in vitro studies showing no effect of VT on either cytokine release from isolated peritoneal macrophages, or migratory capacity of isolated neutrophils. Finally, administration of VT pre-CLP (hazard ratio 0.39 [95% confidence interval 0.19-0.81] P < 0.001) and post-CLP reduced mortality in septic mice (HR 0.22 [95% CI 0.06-0.83] P < 0.05). Conclusions: We provide proof of principle in support of the efficacious use of PEGylated VT, a drug-like Tie2 receptor agonist, to counteract microvascular endothelial barrier dysfunction and reduce mortality in a clinically relevant murine sepsis model. Further studies are needed to pave the road for clinical application of this therapeutic concept