Gßγ directly modulates vesicle fusion by competing with synaptotagmin for binding to neuronal SNARE proteins embedded in membranes.

Gi/o-coupled G protein-coupled receptors can inhibit neurotransmitter release at synapses via multiple mechanisms. In addition to Gßγ-mediated modulation of voltage-gated calcium channels (VGCC), inhibition can also be mediated through the direct interaction of Gßγ subunits with the soluble N-ethylmaleimide attachment protein receptor (SNARE) complex of the vesicle fusion apparatus. Binding studies with soluble SNARE complexes have shown that Gßγ binds to both ternary SNARE complexes, t-SNARE heterodimers, and monomeric SNAREs, competing with synaptotagmin 1(syt1) for binding sites on t-SNARE. However, in secretory cells, Gßγ, SNAREs, and synaptotagmin interact in the lipid environment of a vesicle at the plasma membrane. To approximate this environment, we show that fluorescently labeled Gßγ interacts specifically with lipid-embedded t-SNAREs consisting of full-length syntaxin 1 and SNAP-25B at the membrane, as measured by fluorescence polarization. Fluorescently labeled syt1 undergoes competition with Gßγ for SNARE-binding sites in lipid environments. Mutant Gßγ subunits that were previously shown to be more efficacious at inhibiting Ca2+-triggered exocytotic release than wild-type Gßγ were also shown to bind SNAREs at a higher affinity than wild type in a lipid environment. These mutant Gßγ subunits were unable to inhibit VGCC currents. Specific peptides corresponding to regions on Gß and Gγ shown to be important for the interaction disrupt the interaction in a concentration-dependent manner. In in vitro fusion assays using full-length t- and v-SNAREs embedded in liposomes, Gßγ inhibited Ca2+/synaptotagmin-dependent fusion. Together, these studies demonstrate the importance of these regions for the Gßγ-SNARE interaction and show that the target of Gßγ, downstream of VGCC, is the membrane-embedded SNARE complex.

A Conserved Hydrophobic Core in Gαi1 Regulates G Protein Activation and Release from Activated Receptor.

G protein-coupled receptor-mediated heterotrimeric G protein activation is a major mode of signal transduction in the cell. Previously, we and other groups reported that the α5 helix of Gαi1, especially the hydrophobic interactions in this region, plays a key role during nucleotide release and G protein activation. To further investigate the effect of this hydrophobic core, we disrupted it in Gαi1 by inserting 4 alanine amino acids into the α5 helix between residues Gln(333) and Phe(334) (Ins4A). This extends the length of the α5 helix without disturbing the ß6-α5 loop interactions. This mutant has high basal nucleotide exchange activity yet no receptor-mediated activation of nucleotide exchange. By using structural approaches, we show that this mutant loses critical hydrophobic interactions, leading to significant rearrangements of side chain residues His(57), Phe(189), Phe(191), and Phe(336); it also disturbs the rotation of the α5 helix and the π-π interaction between His(57) and Phe(189) In addition, the insertion mutant abolishes G protein release from the activated receptor after nucleotide binding. Our biochemical and computational data indicate that the interactions between α5, α1, and ß2-ß3 are not only vital for GDP release during G protein activation, but they are also necessary for proper GTP binding (or GDP rebinding). Thus, our studies suggest that this hydrophobic interface is critical for accurate rearrangement of the α5 helix for G protein release from the receptor after GTP binding.

A conserved phenylalanine as a relay between the α5 helix and the GDP binding region of heterotrimeric Gi protein α subunit.

G protein activation by G protein-coupled receptors is one of the critical steps for many cellular signal transduction pathways. Previously, we and other groups reported that the α5 helix in the G protein α subunit plays a major role during this activation process. However, the precise signaling pathway between the α5 helix and the guanosine diphosphate (GDP) binding pocket remains elusive. Here, using structural, biochemical, and computational techniques, we probed different residues around the α5 helix for their role in signaling. Our data showed that perturbing the Phe-336 residue disturbs hydrophobic interactions with the ß2-ß3 strands and α1 helix, leading to high basal nucleotide exchange. However, mutations in ß strands ß5 and ß6 do not perturb G protein activation. We have highlighted critical residues that leverage Phe-336 as a relay. Conformational changes are transmitted starting from Phe-336 via ß2-ß3/α1 to Switch I and the phosphate binding loop, decreasing the stability of the GDP binding pocket and triggering nucleotide release. When the α1 and α5 helices were cross-linked, inhibiting the receptor-mediated displacement of the C-terminal α5 helix, mutation of Phe-336 still leads to high basal exchange rates. This suggests that unlike receptor-mediated activation, helix 5 rotation and translocation are not necessary for GDP release from the α subunit. Rather, destabilization of the backdoor region of the Gα subunit is sufficient for triggering the activation process.

Reovirus-mediated induction of ADAR1 (p150) minimally alters RNA editing patterns in discrete brain regions.

Transcripts encoding ADAR1, a double-stranded, RNA-specific adenosine deaminase involved in the adenosine-to-inosine (A-to-I) editing of mammalian RNAs, can be alternatively spliced to produce an interferon-inducible protein isoform (p150) that is up-regulated in both cell culture and in vivo model systems in response to pathogen or interferon stimulation. In contrast to other tissues, p150 is expressed at extremely low levels in the brain and it is unclear what role, if any, this isoform may play in the innate immune response of the central nervous system (CNS) or whether the extent of editing for RNA substrates critical for CNS function is affected by its induction. To investigate the expression of ADAR1 isoforms in response to viral infection and subsequent alterations in A-to-I editing profiles for endogenous ADAR targets, we used a neurotropic strain of reovirus to infect neonatal mice and quantify A-to-I editing in discrete brain regions using a multiplexed, high-throughput sequencing strategy. While intracranial injection of reovirus resulted in a widespread increase in the expression of ADAR1 (p150) in multiple brain regions and peripheral organs, significant changes in site-specific A-to-I conversion were quite limited, suggesting that steady-state levels of p150 expression are not a primary determinant for modulating the extent of editing for numerous ADAR targets in vivo.

A Systematic Review and Meta-Analysis of the Management of Gallstone Cholecystitis and Common Biliary Duct Stones to Reduce the Incidence of Complications in Elderly Patients.

As the age increases particularly above the age of 50 years, there is a significantly higher risk of developing gallstone-related complications especially cholecystitis and common bile duct stones with its associated consequences. Complications that arise after surgical operations for cholecystitis have been reported to have negative impacts on senior patients. These effects include a higher rate of complications, a longer hospital stay, higher expenditures, and decreased patient satisfaction. Therefore, finding the most effective treatment for cholecystitis in older patients is still a challenge. The aim of the study was carried out in order to identify many approaches that can be taken in the treatment of cholecystitis and stones in the common bile duct in older patients. A search was conducted through Medline (PubMed), EMBASE, ProQuest, and Cochrane using relevant Medical Subject Heading (MeSH) terms and keywords (elderly, age over 50, cholecystitis, bile duct stones, cholecystectomy, ERCP, surgical, conservative management, and open). The searches were limited to studies on elderly individuals over 50 who had cholecystectomy and endoscopic retrograde cholangiopancreatography between January 2000 and December 2022. The meta-analysis used the Mantel-Haenszel odds ratio (MHOR) and 95% confidence interval (CI). Aries Systems Corporation's Editorial Manager® (Aries Systems Corporation, North Andover, USA) and ProduXion Manager® (Aries Systems Corporation, North Andover, USA) facilitated the study. Out of 102 citations, 39 studies were selected for further study. After that, 18 studies were eliminated, leaving 21 for meta-analysis. The study found a protective risk of cholecystitis in cholecystectomy patients (MHOR = 0.16; 95%, CI = 0.10 to 0.25; p 0.001). Developing cholecystitis was substantially lower in early cholecystectomy patients (MHOR = 0.16; 95%, CI = 0.10 to 0.25; p 0.001). There was no significant difference in cholecystitis risk between open and laparoscopic surgery (MHOR = 0.65; 95%, CI = 0.41 to 1.04; p 0.07). Cholecystectomy performed at an earlier stage protects elderly patients from developing recurrent cholecystitis. In contrast to late cholecystitis, in which the patient would experience several attacks of cholecystitis, early cholecystectomy protects against the recurrence of the condition.

Linking receptor activation to changes in Sw I and II of Gα proteins.

G-protein coupled receptors catalyze nucleotide exchange on G proteins, which results in subunit dissociation and effector activation. In the recent ß2AR-Gs structure, portions of Switch I and II of Gα are not fully elucidated. We paired fluorescence studies of receptor-Gαi interactions with the ß2AR-Gs and other Gi structures to investigate changes in Switch I and II during receptor activation and GTP binding. The ß2/ß3 loop containing Leu194 of Gαi is located between Switches I and II, in close proximity to IC2 of the receptor and the C-terminus of Gα, thus providing an allosteric connection between these Switches and receptor activation. We compared the environment of residues in myristoylated Gαi proteins in the heterotrimer to that upon receptor activation and subsequent GTP binding. Upon receptor activation, residues in both Switch regions are less solvent-exposed, as compared to the heterotrimer. Upon GTPγS binding, the environment of several residues in Switch I resemble the receptor-bound state, while Switch II residues display effects on their environment which are consistent with their role in GTP binding and Gßγ dissociation. The ability to merge available crystal structures with solution studies is a powerful tool to gain insight into conformational changes associated with receptor-mediated Gi protein activation.

Myristoylation exerts direct and allosteric effects on Gα conformation and dynamics in solution.

Coupling of heterotrimeric G proteins to activated G protein-coupled receptors results in nucleotide exchange on the Gα subunit, which in turn decreases its affinity for both Gßγ and activated receptors. N-Terminal myristoylation of Gα subunits aids in membrane localization of inactive G proteins. Despite the presence of the covalently attached myristoyl group, Gα proteins are highly soluble after GTP binding. This study investigated factors facilitating the solubility of the activated, myristoylated protein. In doing so, we also identified myristoylation-dependent differences in regions of Gα known to play important roles in interactions with receptors, effectors, and nucleotide binding. Amide hydrogen-deuterium exchange and site-directed fluorescence of activated proteins revealed a solvent-protected amino terminus that was enhanced by myristoylation. Furthermore, fluorescence quenching confirmed that the myristoylated amino terminus is in proximity to the Switch II region in the activated protein. Myristoylation also stabilized the interaction between the guanine ring and the base of the α5 helix that contacts the bound nucleotide. The allosteric effects of myristoylation on protein structure, function, and localization indicate that the myristoylated amino terminus of Gα(i) functions as a myristoyl switch, with implications for myristoylation in the stabilization of nucleotide binding and in the spatial regulation of G protein signaling.

In situ anomalous small-angle X-ray scattering studies of platinum nanoparticle fuel cell electrocatalyst degradation.

Polymer electrolyte fuel cells (PEFCs) are a promising high-efficiency energy conversion technology, but their cost-effective implementation, especially for automotive power, has been hindered by degradation of the electrochemically active surface area (ECA) of the Pt nanoparticle electrocatalysts. While numerous studies using ex situ post-mortem techniques have provided insight into the effect of operating conditions on ECA loss, the governing mechanisms and underlying processes are not fully understood. Toward the goal of elucidating the electrocatalyst degradation mechanisms, we have followed Pt nanoparticle growth during potential cycling of the electrocatalyst in an aqueous acidic environment using in situ anomalous small-angle X-ray scattering (ASAXS). ASAXS patterns were analyzed to obtain particle size distributions (PSDs) of the Pt nanoparticle electrocatalysts at periodic intervals during the potential cycling. Oxide coverages reached under the applied potential cycling protocols were both calculated and determined experimentally. Changes in the PSD, mean diameter, and geometric surface area identify the mechanism behind Pt nanoparticle coarsening in an aqueous environment. Over the first 80 potential cycles, the dominant Pt surface area loss mechanism when cycling to 1.0-1.1 V was found to be preferential dissolution or loss of the smallest particles with varying extents of reprecipitation of the dissolved species onto existing particles, resulting in particle growth, depending on potential profile. Correlation of ASAXS-determined particle growth with both calculated and voltammetrically determined oxide coverages demonstrates that the oxide coverage is playing a key role in the dissolution process and in the corresponding growth of the mean Pt nanoparticle size and loss of ECA. This understanding potentially reduces the complex changes in PSD and ECA resulting from various voltage profiles to a response dependent on oxide coverage.

Exploring the Promise of Multifunctional "Zintl-Phase-Forming" Electrolytes for Si-Based Full Cells.

Li-M-Si ternary Zintl phases have gained attention recently due to their high structural stability, which can improve the cycling stability compared to a bulk Si electrode. Adding multivalent cation salts (such as Mg2+ and Ca2+) in the electrolyte was proven to be a simple way to form Li-M-Si ternary phases in situ in Si-based Li-ion cells. To explore the promise of Zintl-phase-forming electrolytes, we systematically investigated their application in pouch cells via electrochemical and multiscale postmortem analysis. The introduction of multivalent cations, such as Mg2+, during charging can form LixMySi ternary phases. They can stabilize Si anions and reduce side reactions with electrolyte, improving the bulk stability. More importantly, Mg2+ and Ca2+ incorporate into interfacial side reactions and generate inorganic-rich solid-electrolyte interphase, thus enhancing the interfacial stability. Therefore, the full cells with Zintl-phase-forming electrolytes achieve higher capacity retentions at the C/3 rate after 100 cycles, compared to a baseline electrolyte. Additionally, strategies for mitigating the electrode-level fractures of Si were evaluated to make the best use of Zintl-phase-forming electrolytes. This work highlights the significance of synergistic impact of multifunctional additives to stabilize both bulk and interface chemistry in high-energy Si anode materials for Li-ion batteries.

Single-centre experience of an early cannulation graft for haemodialysis access.

INTRODUCTION: As the demographics of the population changes, increasing challenges are being faced in providing reliable access for dialysis. This article reports on the outcomes from the largest series to date using the early cannulation graft Flixene in a single centre. METHODS: Between May 2012 and March 2018, 141 Flixene grafts were placed for dialysis access. The outcomes of the arteriovenous grafts were reviewed retrospectively from electronically held records and imaging. RESULTS: In 75 patients, placement of Flixene graft was performed on an emergency basis and in 66 patients on a planned elective list. The 12-month primary, assisted primary and secondary patency rates were 48.7%, 56.6% and 83.6%, respectively. Eight (5.7%) patients developed infections of the graft during the follow-up period. CONCLUSION: In our experience, we have found the use of the early cannulation graft Flixene to be safe with a low complication rate and favourable patency rates. We believe these early cannulation grafts provide a useful addition for vascular access surgeons preventing the use of tunnelled lines and providing more flexibility in the timing of placing a graft for dialysis.

In situ small-angle X-ray scattering observation of Pt catalyst particle growth during potential cycling.

A unique type of inorganic-organic hybrid semiconductor bulk material is capable of emitting direct white light. Their photoluminescence properties can be tuned precisely and systematically by modifying structures and composition. They could be used as a single-material light-emitting source in high efficiency white-light-emitting diodes.