Predicting overt hepatic encephalopathy after TIPS: Value of three minimal hepatic encephalopathy tests.

Background & Aims: Hepatic encephalopathy (HE) is a frequent and severe complication in patients after transjugular intrahepatic portosystemic shunt (TIPS) insertion. However, risk factors for post-TIPS HE remain poorly defined. Minimal HE (mHE) is a well-known risk factor for overt HE in patients with cirrhosis without TIPS. We aimed to evaluate three tools frequently used for diagnosing mHE for their dynamic changes and their predictive value for overt HE after TIPS. Methods: We prospectively recruited 84 consecutive patients before TIPS insertion and monitored them for 180 days for post-TIPS HE. Before TIPS insertion, the patients underwent the portosystemic encephalopathy (PSE) syndrome test, the animal naming test (ANT), and the critical flicker frequency (CFF). Patients were retested after TIPS insertion. Results: The majority of patients were male (67.9%), and the predominant indication for TIPS was refractory ascites (75%). Median age was 59 years, model for end-stage liver disease score was 12, and 66.3%, 64.6%, and 28.4% patients had evidence for mHE according to the PSE syndrome test, ANT, and CFF, respectively. Overall, 25 patients developed post-TIPS HE within 180 days after TIPS insertion. Post-TIPS incidence of overt HE was 22.2, 28.6, 45.5, and 55.6% in those with no, one, two, and three pathological tests at baseline, respectively. However, none of the three tests was significantly associated with post-TIPS HE. Of note, mean performance in all tests remained stable over time after TIPS insertion. Conclusions: PSE syndrome test, ANT and CFF, which are frequently used for diagnosing mHE have limited value for predicting HE after TIPS insertion. We could not find evidence that TIPS insertion leads to a psychometric decline in the long term. Impact and implications: This prospective observational study compared three diagnostic tests for mHE and showed the limited value of these tests for predicting overt HE in patients with cirrhosis undergoing TIPS insertion. In addition, the results suggest that cognitive performance generally remains stable after TIPS insertion. These results are important for physicians and researchers involved in the management of patients with cirrhosis undergoing TIPS procedures. The study's findings serve as a starting point for further investigations on the development of more effective strategies for predicting and managing post-TIPS HE. Clinical trial number: ClinicalTrials.gov NCT04801290.

Integrative dynamic structural biology unveils conformers essential for the oligomerization of a large GTPase.

Guanylate binding proteins (GBPs) are soluble dynamin-like proteins that undergo a conformational transition for GTP-controlled oligomerization and disrupt membranes of intracellular parasites to exert their function as part of the innate immune system of mammalian cells. We apply neutron spin echo, X-ray scattering, fluorescence, and EPR spectroscopy as techniques for integrative dynamic structural biology to study the structural basis and mechanism of conformational transitions in the human GBP1 (hGBP1). We mapped hGBP1's essential dynamics from nanoseconds to milliseconds by motional spectra of sub-domains. We find a GTP-independent flexibility of the C-terminal effector domain in the µs-regime and resolve structures of two distinct conformers essential for an opening of hGBP1 like a pocket knife and for oligomerization. Our results on hGBP1's conformational heterogeneity and dynamics (intrinsic flexibility) deepen our molecular understanding relevant for its reversible oligomerization, GTP-triggered association of the GTPase-domains and assembly-dependent GTP-hydrolysis.

Prickle promotes the formation and maintenance of glutamatergic synapses by stabilizing the intercellular planar cell polarity complex.

Whether there exists a common signaling mechanism that assembles all glutamatergic synapses is unknown. We show here that knocking out Prickle1 and Prickle2 reduced the formation of the PSD-95­positive glutamatergic synapses in the hippocampus and medial prefrontal cortex in postnatal development by 70­80%. Prickle1 and Prickle2 double knockout in adulthood lead to the disassembly of 70 to 80% of the postsynaptic-density(PSD)-95­positive glutamatergic synapses. PSD-95­positive glutamatergic synapses in the hippocampus of Prickle2E8Q/E8Q mice were reduced by 50% at postnatal day 14. Prickle2 promotes synapse formation by antagonizing Vangl2 and stabilizing the intercellular complex of the planar cell polarity (PCP) components, whereas Prickle2 E8Q fails to do so. Coculture experiments show that the asymmetric PCP complexes can determine the presynaptic and postsynaptic polarity. In summary, the PCP components regulate the assembly and maintenance of a large number of glutamatergic synapses and specify the direction of synaptic transmission.

Farnesylation of human guanylate-binding protein 1 as safety mechanism preventing structural rearrangements and uninduced dimerization.

Human guanylate-binding protein 1 (hGBP1) belongs to the family of dynamin-like proteins and is activated by addition of nucleotides, leading to protein oligomerization and stimulated GTPase activity. In vivo, hGBP1 is post-translationally modified by attachment of a farnesyl group yielding farn-hGBP1. In this study, hydrodynamic differences in farn-hGBP1 and unmodified hGBP1 were investigated using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and analytical size-exclusion chromatography (SEC). In addition, we performed small-angle X-ray scattering (SAXS) experiments coupled with a SEC setup (SEC-SAXS) to investigate structural properties of nonmodified hGBP1 and farn-hGBP1 in solution. SEC-SAXS measurements revealed that farnesylation keeps hGBP1 in its inactive monomeric and crystal-like conformation in nucleotide-free solution, whereas unmodified hGBP1 forms a monomer-dimer equilibrium both in the inactive ground state in nucleotide-free solution as well as in the activated state that is trapped by addition of the nonhydrolysable GTP analogue GppNHp. Nonmodified hGBP1 is structurally perturbed as compared to farn-hGBP. In particular, GppNHp binding leads to large structural rearrangements and higher conformational flexibility of the monomer and the dimer. Structural changes observed in the nonmodified protein are prerequisites for further oligomer assemblies of farn-hGBP1 that occur in the presence of nucleotides. DATABASE: All SEC-SAXS data, corresponding fits to the data and structural models are deposited in the Small Angle Scattering Biological Data Bank [SASBDB (Nucleic Acids Res, 43, 2015, D357)] with project IDs: SASDEE8, SASDEF8, SASDEG8, SASDEH8, SASDEJ8, SASDEK8, SASDEL8 and SASDEM8.

Combined Small-Angle X-ray and Neutron Scattering Restraints in Molecular Dynamics Simulations.

Small-angle X-ray and small-angle neutron scattering (SAXS/SANS) provide unique structural information on biomolecules and their complexes in solution. SANS may provide multiple independent data sets by means of contrast variation experiments, that is, by measuring at different D2O concentrations and different perdeuteration conditions of the biomolecular complex. However, even the combined data from multiple SAXS/SANS sets is by far insufficient to define all degrees of freedom of a complex, leading to a significant risk of overfitting when refining biomolecular structures against SAXS/SANS data. Hence, to control against overfitting, the low-information SAXS/SANS data must be complemented by accurate physical models, and, if possible, refined models should be cross-validated against independent data not used during the refinement. We present a method for refining atomic biomolecular structures against multiple sets of SAXS and SANS data using all-atom molecular dynamics simulations. Using the protein citrate synthase and the protein/RNA complex Sxl-Unr-msl2 mRNA as test cases, we demonstrate how multiple SAXS and SANS sets may be used for refinement and cross-validation, thereby excluding overfitting during refinement. For the Sxl-Unr-msl2 complex, we find that perdeuteration of the Unr domain leads to a unique, slightly compacted conformation, whereas other perdeuteration conditions lead to similar solution conformations compared to the nondeuterated state. In line with our previous method for predicting SAXS curves, SANS curves were predicted with explicit-solvent calculations, taking atomic models for both the hydration layer and the excluded solvent into account, thereby avoiding the use of solvent-related fitting parameters and solvent-reduced neutron scattering lengths. We expect the method to be useful for deriving and validating solution structures of biomolecules and soft-matter complexes, and for critically assessing whether multiple SAXS and SANS sets are mutually compatible.

Photochemical Properties of the Red-shifted Channelrhodopsin Chrimson.

Color-tuned variants of channelrhodopsins allow for selective optogenetic manipulation of different host cell populations. Chrimson is the channelrhodopsin with the longest wavelength absorbance maximum. We characterize its photochemical properties at different pH values corresponding to two protonation states of the counterion for the protonated Schiff base. Both states will lead to a functional channel opening, but the route is different as reflected in the photochemical states observed spectroscopically. The light-induced isomerization kinetics change with the local electrostatic environment, becoming faster with the presence of an anionic counterion. The spectral effect is stronger on the ground-state energy surface. From the excited state, a bifurcated pathway leads to the electronic ground state resulting in a pronounced excitation wavelength dependence. The subsequent steps in the photocycles at pH 6 and pH 9.5 differ in the accumulation of states with a protonated and deprotonated Schiff base, respectively, that can be correlated with the open channel. Therefore, different protonation states are preserved in the open and the initial states. Chrimson's photocycle at alkaline pH shows features observed in other rhodopsins without an internal proton donor to the Schiff base, but it accumulates an intermediate with an even longer lifetime reflecting slow recovery of the initial state.