Accelerated generation of gene-engineered monoclonal CHO cell lines using FluidFM nanoinjection and CRISPR/Cas9.

Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow. Here, in this proof-of-concept (PoC) study, we present a novel approach coined CellEDIT to engineer CHO cells by intranuclear delivery of the CRISPR components to single cells using the FluidFM technology. Co-injection of CRISPR system targeting BAX, DHFR, and FUT8 directly into the nucleus of single cells, enabled us to generate triple knockout CHO-K1 cell lines within a short time frame. The proposed technique assures the origin of monoclonality without the requirement of limiting dilution, cell sorting or positive selection. Furthermore, the approach is compatible to develop both single and multiple knockout clones (FUT8, BAX, and DHFR) in CHO cells. Further analyses on single and multiple knockout clones confirmed the targeted genetic disruption and altered protein expression. The knockout CHO-K1 clones showed the persistence of gene editing during the subsequent passages, compatible with serum free chemically defined media and showed equivalent transgene expression like parental clone.

Impact of glycan nature on structure and viscoelastic properties of glycopeptide hydrogels.

Mucus is a complex biological hydrogel that acts as a barrier for almost everything entering or exiting the body. It is therefore of emerging interest for biomedical and pharmaceutical applications. Besides water, the most abundant components are the large and densely glycosylated mucins, glycoproteins of up to 20 MDa and carbohydrate content of up to 80 wt%. Here, we designed and explored a library of glycosylated peptides to deconstruct the complexity of mucus. Using the well-characterized hFF03 coiled-coil system as a hydrogel-forming peptide scaffold, we systematically probed the contribution of single glycans to the secondary structure as well as the formation and viscoelastic properties of the resulting hydrogels. We show that glycan-decoration does not affect α-helix and coiled-coil formation while it alters gel stiffness. By using oscillatory macrorheology, dynamic light scattering microrheology, and fluorescence lifetime-based nanorheology, we characterized the glycopeptide materials over several length scales. Molecular simulations revealed that the glycosylated linker may extend into the solvent, but more frequently interacts with the peptide, thereby likely modifying the stability of the self-assembled fibers. This systematic study highlights the interplay between glycan structure and hydrogel properties and may guide the development of synthetic mucus mimetics.

Transposition of HOPPLA in siRNA-deficient plants suggests a limited effect of the environment on retrotransposon mobility in Brachypodium distachyon.

Long terminal repeat retrotransposons (LTR-RTs) are powerful mutagens regarded as a major source of genetic novelty and important drivers of evolution. Yet, the uncontrolled and potentially selfish proliferation of LTR-RTs can lead to deleterious mutations and genome instability, with large fitness costs for their host. While population genomics data suggest that an ongoing LTR-RT mobility is common in many species, the understanding of their dual role in evolution is limited. Here, we harness the genetic diversity of 320 sequenced natural accessions of the Mediterranean grass Brachypodium distachyon to characterize how genetic and environmental factors influence plant LTR-RT dynamics in the wild. When combining a coverage-based approach to estimate global LTR-RT copy number variations with mobilome-sequencing of nine accessions exposed to eight different stresses, we find little evidence for a major role of environmental factors in LTR-RT accumulations in B. distachyon natural accessions. Instead, we show that loss of RNA polymerase IV (Pol IV), which mediates RNA-directed DNA methylation in plants, results in high transcriptional and transpositional activities of RLC_BdisC024 (HOPPLA) LTR-RT family elements, and that these effects are not stress-specific. This work supports findings indicating an ongoing mobility in B. distachyon and reveals that host RNA-directed DNA methylation rather than environmental factors controls their mobility in this wild grass model.

Thermal isomerization rates in retinal analogues using Ab-Initio molecular dynamics.

For a detailed understanding of chemical processes in nature and industry, we need accurate models of chemical reactions in complex environments. While Eyring transition state theory is commonly used for modeling chemical reactions, it is most accurate for small molecules in the gas phase. A wide range of alternative rate theories exist that can better capture reactions involving complex molecules and environmental effects. However, they require that the chemical reaction is sampled by molecular dynamics simulations. This is a formidable challenge since the accessible simulation timescales are many orders of magnitude smaller than typical timescales of chemical reactions. To overcome these limitations, rare event methods involving enhanced molecular dynamics sampling are employed. In this work, thermal isomerization of retinal is studied using tight-binding density functional theory. Results from transition state theory are compared to those obtained from enhanced sampling. Rates obtained from dynamical reweighting using infrequent metadynamics simulations were in close agreement with those from transition state theory. Meanwhile, rates obtained from application of Kramers' rate equation to a sampled free energy profile along a torsional dihedral reaction coordinate were found to be up to three orders of magnitude higher. This discrepancy raises concerns about applying rate methods to one-dimensional reaction coordinates in chemical reactions.

How Chromophore Labels Shape the Structure and Dynamics of a Peptide Hydrogel.

Biocompatible and functionalizable hydrogels have a wide range of (potential) medicinal applications. The hydrogelation process, particularly for systems with very low polymer weight percentages (<1 wt %), remains poorly understood, making it challenging to predict the self-assembly of a given molecular building block into a hydrogel. This severely hinders the rational design of self-assembled hydrogels. In this study, we demonstrate the impact of an N-terminal group on the self-assembly and rheology of the peptide hydrogel hFF03 (hydrogelating, fibril forming peptide 03) using molecular dynamics simulations, oscillatory shear rheology, and circular dichroism spectroscopy. We find that the chromophore and even its specific regioisomers have a significant influence on the microscopic structure and dynamics of the self-assembled fibril, and on the macroscopic mechanical properties. This is because the chromophore influences the possible salt bridges, which form and stabilize the fibril formation. Furthermore, we find that the solvation shell fibrils by itself cannot explain the viscoelasticity of hFF03 hydrogels. Our atomistic model of the hFF03 fibril formation enables a more rational design of these hydrogels. In particular, altering the N-terminal chromophore emerges as a design strategy to tune the mechanic properties of these self-assembled peptide hydrogels.

Biosynthetic incorporation of fluorinated amino acids into the nonribosomal peptide gramicidin S.

Fluorine is a key element in medicinal chemistry, as it can significantly enhance the pharmacological properties of drugs. In this study, we aimed to biosynthetically produce fluorinated analogues of the antimicrobial cyclic decapeptide gramicidin S (GS). However, our results show that the A-domain of the NRPS module GrsA rejects 4-fluorinated analogues of its native substrate Phe due to an interrupted T-shaped aromatic interaction in the binding pocket. We demonstrate that GrsA mutant W239S improves the incorporation of 4-fluorinated Phe into GS both in vitro and in vivo. Our findings provide new insights into the behavior of NRPSs towards fluorinated amino acids and strategies for the engineered biosynthesis of fluorinated peptides.

Optimizing molecular potential models by imposing kinetic constraints with path reweighting.

Empirical force fields employed in molecular dynamics simulations of complex systems are often optimized to reproduce experimentally determined structural and thermodynamic properties. In contrast, experimental knowledge about the interconversion rates between metastable states in such systems is hardly ever incorporated in a force field due to a lack of an efficient approach. Here, we introduce such a framework based on the relationship between dynamical observables, such as rate constants, and the underlying molecular model parameters using the statistical mechanics of trajectories. Given a prior ensemble of molecular dynamics trajectories produced with imperfect force field parameters, the approach allows for the optimal adaption of these parameters such that the imposed constraint of equally predicted and experimental rate constant is obeyed. To do so, the method combines the continuum path ensemble maximum caliber approach with path reweighting methods for stochastic dynamics. When multiple solutions are found, the method selects automatically the combination that corresponds to the smallest perturbation of the entire path ensemble, as required by the maximum entropy principle. To show the validity of the approach, we illustrate the method on simple test systems undergoing rare event dynamics. Next to simple 2D potentials, we explore particle models representing molecular isomerization reactions and protein-ligand unbinding. Besides optimal interaction parameters, the methodology gives physical insights into what parts of the model are most sensitive to the kinetics. We discuss the generality and broad implications of the methodology.

[Malnutrition in internal medicine : Screening, assessment and importance]. / Mangelernährung in der Inneren Medizin : Screening, Assessment und Bedeutung.

Disease-related malnutrition has a strong influence on the further course of the disease and mortality, especially in chronically ill patients. In recent years it could be shown in large randomized studies that an individual nutrition therapy could significantly and relevantly improve the clinical outcome of patients in internal medicine with a risk of malnutrition, both in hospital and in aftercare. Therefore, due to the increasing proportion of multimorbid patients the significance of malnutrition and its treatment is becoming increasingly more important in the practice and in research. Nutritional medicine should nowadays be considered as an effective and integral component of a holistic treatment in internal medicine; however, further research is necessary in order to investigate new nutritional biomarkers and for a better integration of an evidence-based personalized nutritional medicine into routine clinical practice.

Inflammation and Nutrition: Friend or Foe?

The importance of the interplay between inflammation and nutrition has generated much interest in recent times. Inflammation has been identified as a key driver for disease-related malnutrition, leading to anorexia, reduced food intake, muscle catabolism, and insulin resistance, which are stimulating a catabolic state. Interesting recent data suggest that inflammation also modulates the response to nutritional treatment. Studies have demonstrated that patients with high inflammation show no response to nutritional interventions, while patients with lower levels of inflammation do. This may explain the contradictory results of nutritional trials to date. Several studies of heterogeneous patient populations, or in the critically ill or advanced cancer patients, have not found significant benefits on clinical outcome. Vice versa, several dietary patterns and nutrients with pro- or anti-inflammatory properties have been identified, demonstrating that nutrition influences inflammation. Within this review, we summarize and discuss recent advances in both the role of inflammation in malnutrition and the effect of nutrition on inflammation.

CommonNNClustering─A Python Package for Generic Common-Nearest-Neighbor Clustering.

Density-based clustering procedures are widely used in a variety of data science applications. Their advantage lies in the capability to find arbitrarily shaped and sized clusters and robustness against outliers. In particular, they proved effective in the analysis of molecular dynamics simulations, where they serve to identify relevant, low-energetic molecular conformations. As such, they can provide a convenient basis for the construction of kinetic (core-set) Markov-state models. Here we present the open-source Python project CommonNNClustering, which provides an easy-to-use and efficient reimplementation of the common-nearest-neighbor (CommonNN) method. The package provides functionalities for hierarchical clustering and an evaluation of the results. We put our emphasis on a generic API design to keep the implementation flexible and open for customization.

Water Network in the Binding Pocket of Fluorinated BPTI-Trypsin Complexes─Insights from Simulation and Experiment.

Structural waters in the S1 binding pocket of ß-trypsin are critical for the stabilization of the complex of ß-trypsin with its inhibitor bovine pancreatic trypsin inhibitor (BPTI). The inhibitor strength of BPTI can be modulated by replacing the critical lysine residue at the P1 position by non-natural amino acids. We study BPTI variants in which the critical Lys15 in BPTI has been replaced by α-aminobutyric acid (Abu) and its fluorinated derivatives monofluoroethylglycine (MfeGly), difluoroethylglycine (DfeGly), and trifluoroethylglycine (TfeGly). We investigate the hypothesis that additional water molecules in the binding pocket can form specific noncovalent interactions with the fluorinated side chains and thereby act as an extension of the inhibitors. We report potentials of mean force (PMF) of the unbinding process for all four complexes and enzyme activity inhibition assays. Additionally, we report the protein crystal structure of the Lys15MfeGly-BPTI-ß-trypsin complex (pdb: 7PH1). Both experimental and computational data show a stepwise increase in inhibitor strength with increasing fluorination of the Abu side chain. The PMF additionally shows a minimum for the encounter complex and an intermediate state just before the bound state. In the bound state, the computational analysis of the structure and dynamics of the water molecules in the S1 pocket shows a highly dynamic network of water molecules that does not indicate a rigidification or stabilizing trend in regard to energetic properties that could explain the increase in inhibitor strength. The analysis of the energy and the entropy of the water molecules in the S1 binding pocket using grid inhomogeneous solvation theory confirms this result. Overall, fluorination systematically changes the binding affinity, but the effect cannot be explained by a persistent water network in the binding pocket. Other effects, such as the hydrophobicity of fluorinated amino acids and the stability of the encounter complex as well as the additional minimum in the potential of mean force in the bound state, likely influence the affinity more directly.

The occurrence of ansamers in the synthesis of cyclic peptides.

α-Amanitin is a bicyclic octapeptide composed of a macrolactam with a tryptathionine cross-link forming a handle. Previously, the occurrence of isomers of amanitin, termed atropisomers has been postulated. Although the total synthesis of α-amanitin has been accomplished this aspect still remains unsolved. We perform the synthesis of amanitin analogs, accompanied by in-depth spectroscopic, crystallographic and molecular dynamics studies. The data unambiguously confirms the synthesis of two amatoxin-type isomers, for which we propose the term ansamers. The natural structure of the P-ansamer can be ansa-selectively synthesized using an optimized synthetic strategy. We believe that the here described terminology does also have implications for many other peptide structures, e.g. norbornapeptides, lasso peptides, tryptorubins and others, and helps to unambiguously describe conformational isomerism of cyclic peptides.

GROMACS Stochastic Dynamics and BAOAB Are Equivalent Configurational Sampling Algorithms.

Two of the most widely used Langevin integrators for molecular dynamics simulations are the GROMACS Stochastic Dynamics (GSD) integrator and the splitting method BAOAB. In this letter, we show that the GROMACS Stochastic Dynamics integrator is equal to the less frequently used splitting method BAOA. It immediately follows that GSD and BAOAB sample the same configurations and have the same high configurational accuracy. Our numerical results indicate that GSD/BAOA has higher kinetic accuracy than BAOAB.

A Formylglycine-Peptide for the Site-Directed Identification of Phosphotyrosine-Mimetic Fragments.

Discovery of protein-binding fragments for precisely defined binding sites is an unmet challenge to date. Herein, formylglycine is investigated as a molecular probe for the sensitive detection of fragments binding to a spatially defined protein site . Formylglycine peptide 3 was derived from a phosphotyrosine-containing peptide substrate of protein tyrosine phosphatase PTP1B by replacing the phosphorylated amino acid with the reactive electrophile. Fragment ligation with formylglycine occurred in situ in aqueous physiological buffer. Structures and kinetics were validated by NMR spectroscopy. Screening and hit validation revealed fluorinated and non-fluorinated hit fragments being able to replace the native phosphotyrosine residue. The formylglycine probe identified low-affinity fragments with high spatial resolution as substantiated by molecular modelling. The best fragment hit, 4-amino-phenyl-acetic acid, was converted into a cellularly active, nanomolar inhibitor of the protein tyrosine phosphatase SHP2.

Fluorine-induced polarity increases inhibitory activity of BPTI towards chymotrypsin.

Substituting the P1 position in bovine pancreatic trypsin inhibitor (BPTI) is known to heavily influence its inhibitory activity towards serine proteases. Side-chain fluorinated aliphatic amino acids have been shown to alter numerous properties of peptides and proteins and thus are of interest in the context of BPTI. In our study, we systematically investigated the site-specific incorporation of non-canonical amino acids into BPTI by microwave-assisted solid-phase peptide synthesis (SPPS). Inhibitor activity of the variants was tested towards the serine protease α-chymotrypsin. We observed enhanced inhibition of two fluorinated BPTIs compared to wild type and hydrocarbon variants. To further investigate the complexes, we performed X-ray structure analysis. Our findings underline the power fluorine offers as a tool in protein engineering to beneficially alter the effects on phenomena as protein-protein interactions.

Experimentally heat-induced transposition increases drought tolerance in Arabidopsis thaliana.

Eukaryotic genomes contain a vast diversity of transposable elements (TEs). Formerly often described as selfish and parasitic DNA sequences, TEs are now recognised as a source of genetic diversity and powerful drivers of evolution. However, because their mobility is tightly controlled by the host, studies experimentally assessing how fast TEs may mediate the emergence of adaptive traits are scarce. We exposed Arabidopsis thaliana high-copy TE lines (hcLines) with up to c. eight-fold increased copy numbers of the heat-responsive ONSEN TE to drought as a straightforward and ecologically highly relevant selection pressure. We provide evidence for increased drought tolerance in five out of the 23 tested hcLines and further pinpoint one of the causative mutations to an exonic insertion of ONSEN in the ribose-5-phosphate-isomerase 2 gene. The resulting loss-of-function mutation caused a decreased rate of photosynthesis, plant size and water consumption. Overall, we show that the heat-induced transposition of a low-copy TE increases phenotypic diversity and leads to the emergence of drought-tolerant individuals in A. thaliana. This is one of the rare empirical examples substantiating the adaptive potential of mobilised stress-responsive TEs in eukaryotes. Our work demonstrates the potential of TE-mediated loss-of-function mutations in stress adaptation.

Target Recognition in Tandem WW Domains: Complex Structures for Parallel and Antiparallel Ligand Orientation in h-FBP21 Tandem WW.

Protein-protein interactions often rely on specialized recognition domains, such as WW domains, which bind to specific proline-rich sequences. The specificity of these protein-protein interactions can be increased by tandem repeats, i.e., two WW domains connected by a linker. With a flexible linker, the WW domains can move freely with respect to each other. Additionally, the tandem WW domains can bind in two different orientations to their target sequences. This makes the elucidation of complex structures of tandem WW domains extremely challenging. Here, we identify and characterize two complex structures of the tandem WW domain of human formin-binding protein 21 and a peptide sequence from its natural binding partner, the core-splicing protein SmB/B'. The two structures differ in the ligand orientation and, consequently, also in the relative orientation of the two WW domains. We analyze and probe the interactions in the complexes by molecular simulations and NMR experiments. The workflow to identify the complex structures uses molecular simulations, density-based clustering, and peptide docking. It is designed to systematically generate possible complex structures for repeats of recognition domains. These structures will help us to understand the synergistic and multivalency effects that generate the astonishing versatility and specificity of protein-protein interactions.

Pentafluorophosphato-Phenylalanines: Amphiphilic Phosphotyrosine Mimetics Displaying Fluorine-Specific Protein Interactions.

Phosphotyrosine residues are essential functional switches in health and disease. Thus, phosphotyrosine biomimetics are crucial for the development of chemical tools and drug molecules. We report here the discovery and investigation of pentafluorophosphato amino acids as novel phosphotyrosine biomimetics. A mild acidic pentafluorination protocol was developed and two PF5 -amino acids were prepared and employed in peptide synthesis. Their structures, reactivities, and fluorine-specific interactions were studied by NMR and IR spectroscopy, X-ray diffraction, and in bioactivity assays. The mono-anionic PF5 motif displayed an amphiphilic character binding to hydrophobic surfaces, to water molecules, and to protein-binding sites, exploiting charge and H-F-bonding interactions. The novel motifs bind 25- to 30-fold stronger to the phosphotyrosine binding site of the protein tyrosine phosphatase PTP1B than the best current biomimetics, as rationalized by computational methods, including molecular dynamics simulations.

Stable Isotopomers of myo-Inositol Uncover a Complex MINPP1-Dependent Inositol Phosphate Network.

The water-soluble inositol phosphates (InsPs) represent a functionally diverse group of small-molecule messengers involved in a myriad of cellular processes. Despite their centrality, our understanding of human InsP metabolism is incomplete because the available analytical toolset to characterize and quantify InsPs in complex samples is limited. Here, we have synthesized and applied symmetrically and unsymmetrically 13C-labeled myo-inositol and inositol phosphates. These probes were utilized in combination with nuclear magnetic resonance spectroscopy (NMR) and capillary electrophoresis mass spectrometry (CE-MS) to investigate InsP metabolism in human cells. The labeling strategy provided detailed structural information via NMR-down to individual enantiomers-which overcomes a crucial blind spot in the analysis of InsPs. We uncovered a novel branch of InsP dephosphorylation in human cells which is dependent on MINPP1, a phytase-like enzyme contributing to cellular homeostasis. Detailed characterization of MINPP1 activity in vitro and in cells showcased the unique reactivity of this phosphatase. Our results demonstrate that metabolic labeling with stable isotopomers in conjunction with NMR spectroscopy and CE-MS constitutes a powerful tool to annotate InsP networks in a variety of biological contexts.

Body composition analysis in patients with acute heart failure: the Scale Heart Failure trial.

AIMS: In this study, we aimed to investigate whether body composition analysis (BCA) derived from bioelectrical impedance vector analysis (BIVA) could be used to monitor the hydration status of patients with acute heart failure (AHF) during intensified diuretic therapy. METHODS AND RESULTS: This observational, single-centre study involved a novel, validated eight-electrode segmental body composition analyser to perform BCA derived from BIVA with an alternating current of 100 µA at frequencies of 5, 7.5, 50, and 75 kHz. The BCA-derived and BIVA-derived parameters were estimated and compared with daily body weight measurements in hospitalized patients with AHF. A total of 867 BCA and BIVA assessments were conducted in 142 patients (56.3% men; age 76.8 ± 10.7 years). Daily changes in total body water (TBW) and extracellular water (ECW) were significantly associated with changes in body weight in 62.2% and 89.1% of all measurements, respectively (range, ±1 kg). Repeated measures correlation coefficients between weight loss and TBW loss resulted with rho 0.43, P < 0.01, confidence interval (CI) [0.36, 0.50] and rho 0.71, P > 0.01, CI [0.67, 0.75] for ECW loss. Between the first and last assessments, the mean weight loss was -2.5 kg, compared with the -2.6 L mean TBW loss and -1.7 L mean ECW loss. BIVA revealed an increase in mean Resistance R and mean Reactance Xc across all frequencies, with the subsequent reduction in body fluid (including corresponding body weight) between the first and last assessments. CONCLUSIONS: Body composition analysis derived from BIVA with a focus on ECW is a promising approach to detect changes in hydration status in patients undergoing intensified diuretic therapy. Defining personalized BIVA reference values using bioelectrical impedance devices is a promising approach to monitor hydration status.