Efficacy of Alkaline Phosphatase in Critically Ill Patients with COVID-19: A Multicentre Investigator-Initiated Double-Blind Randomised Placebo-Controlled Trial.

Background: Efforts to identify therapies to treat hospitalised patients with COVID-19 are being continued. Alkaline phosphatase (AP) dephosphorylates pro-inflammatory adenosine triphosphate (ATP) into anti-inflammatory adenosine.

Methods: In a randomised controlled trial, we investigated the safety and efficacy of AP in patients with SARS-CoV-2 infection admitted to the ICU.

Tibial Nerve Block as Treatment of Chronic Foot Pain.

Background: Minimally invasive therapies can alleviate pain and improve walking in individuals with persistent foot and ankle pain.

Objectives: The current study aimed to define the protracted consequences of tibial nerve blocks with steroids for individuals with persistent foot pain and to investigate the link between the thermography of the plantar foot and the beneficial effect of a tibial nerve block with steroids.

Methods: All patients with chronic foot pain (n = 45) in this cohort underwent a block of the tibial nerve in the Department of Pain Therapy of Pain Clinic De Bilt, Utrecht, Netherlands, within November 2019 to April 2020.

A multimodal pain protocol for treatment of vaso-occlusive crisis in patients with sickle cell disease: Implementation and evaluation.

Objectives: Pain management during a vaso-occlusive crisis (VOC) for patients with sickle cell disease (SCD) remains a major challenge and strongly depends on opioids. We developed a multimodality pain protocol for rapid, opioid-sparing pain treatment of VOC and evaluated its feasibility.

Methods: Patients were included for evaluation if they were ≥18 years, diagnosed with SCD and visited the emergency department (ED) because of VOC between July 2018 and December 2020.

Autoregulation of GPCR signalling through the third intracellular loop.

The third intracellular loop (ICL3) of the G protein-coupled receptor (GPCR) fold is important for the signal transduction process downstream of receptor activation. Despite this, the lack of a defined structure of ICL3, combined with its high sequence divergence among GPCRs, complicates characterization of its involvement in receptor signalling. Previous studies focusing on the β adrenergic receptor (βAR) suggest that ICL3 is involved in the structural process of receptor activation and signalling.

Multimodal regulation of myosin VI ensemble transport by cargo adaptor protein GIPC.

A range of cargo adaptor proteins are known to recruit cytoskeletal motors to distinct subcellular compartments. However, the structural impact of cargo recruitment on motor function is poorly understood. Here, we dissect the multimodal regulation of myosin VI activity through the cargo adaptor GAIP-interacting protein, C terminus (GIPC), whose overexpression with this motor in cancer enhances cell migration.

β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus.

Classical pharmacological models have incorporated an "intrinsic efficacy" parameter to capture system-independent effects of G protein-coupled receptor (GPCR) ligands. However, the nonlinear serial amplification of downstream signaling limits quantitation of ligand intrinsic efficacy. A recent biophysical study has characterized a ligand "molecular efficacy" that quantifies the influence of ligand-dependent receptor conformation on G protein activation.

KIF13A motors are regulated by Rab22A to function as weak dimers inside the cell.

Endocytic recycling is a complex itinerary, critical for many cellular processes. Membrane tubulation is a hallmark of recycling endosomes (REs), mediated by KIF13A, a kinesin-3 family motor. Understanding the regulatory mechanism of KIF13A in RE tubulation and cargo recycling is of fundamental importance but is overlooked.

Kinase inhibitors allosterically disrupt a regulatory interaction to enhance PKCα membrane translocation.

The eukaryotic kinase domain has multiple intrinsically disordered regions whose conformation dictates kinase activity. Small molecule kinase inhibitors (SMKIs) rely on disrupting the active conformations of these disordered regions to inactivate the kinase. While SMKIs are selected for their ability to cause this disruption, the allosteric effects of conformational changes in disordered regions is limited by a lack of dynamic information provided by traditional structural techniques.

ER/K-link-Leveraging a native protein linker to probe dynamic cellular interactions.

ER/K α-helices are a subset of single alpha helical domains, which exhibit unusual stability as isolated protein secondary structures. They adopt an elongated structural conformation, while regulating the frequency of interactions between proteins or polypeptides fused to their ends. Here we review recent advances on the structure, stability and function of ER/K α-helices as linkers (ER/K linkers) in native proteins.

Dynamic multimerization of Dab2-Myosin VI complexes regulates cargo processivity while minimizing cortical actin reorganization.

Myosin VI ensembles on endocytic cargo facilitate directed transport through a dense cortical actin network. Myosin VI is recruited to clathrin-coated endosomes via the cargo adaptor Dab2. Canonically, it has been assumed that the interactions between a motor and its cargo adaptor are stable.

Minute-scale persistence of a GPCR conformation state triggered by non-cognate G protein interactions primes signaling.

Despite the crowded nature of the cellular milieu, ligand-GPCR-G protein interactions are traditionally viewed as spatially and temporally isolated events. In contrast, recent reports suggest the spatial and temporal coupling of receptor-effector interactions, with the potential to diversify downstream responses. In this study, we combine protein engineering of GPCR-G protein interactions with affinity sequestration and photo-manipulation of the crucial Gα C terminus, to demonstrate the temporal coupling of cognate and non-cognate G protein interactions through priming of the GPCR conformation.

Engaging myosin VI tunes motility, morphology and identity in endocytosis.

While unconventional myosins interact with different stages of the endocytic pathway, they are ascribed a transport function that is secondary to the protein complexes that control organelle identity. Endosomes are subject to a dynamic, continuous flux of proteins that control their characteristic properties, including their motility within the cell. Efforts to describe the changes in identity of this compartment have largely focused on the adaptors present on the compartment and not on the motile properties of the compartment itself.

ER/K linked GPCR-G protein fusions systematically modulate second messenger response in cells.

FRET and BRET approaches are well established for detecting ligand induced GPCR-G protein interactions in cells. Currently, FRET/BRET assays rely on co-expression of GPCR and G protein, and hence depend on the stoichiometry and expression levels of the donor and acceptor probes. On the other hand, GPCR-G protein fusions have been used extensively to understand the selectivity of GPCR signaling pathways.

Priming GPCR signaling through the synergistic effect of two G proteins.

Although individual G-protein-coupled receptors (GPCRs) are known to activate one or more G proteins, the GPCR-G-protein interaction is viewed as a bimolecular event involving the formation of a ternary ligand-GPCR-G-protein complex. Here, we present evidence that individual GPCR-G-protein interactions can reinforce each other to enhance signaling through canonical downstream second messengers, a phenomenon we term "GPCR priming." Specifically, we find that the presence of noncognate Gq protein enhances cAMP stimulated by two Gs-coupled receptors, β2-adrenergic receptor (β2-AR) and D dopamine receptor (D-R).

The Role of Regulatory Domains in Maintaining Autoinhibition in the Multidomain Kinase PKCα.

Resolving the conformational dynamics of large multidomain proteins has proven to be a significant challenge. Here we use a variety of techniques to dissect the roles of individual protein kinase Cα (PKCα) regulatory domains in maintaining catalytic autoinhibition. We find that whereas the pseudosubstrate domain is necessary for autoinhibition it is not sufficient.

Calcium Stimulates Self-Assembly of Protein Kinase C α In Vitro.

Protein kinase C α (PKCα) is a nodal regulator in several intracellular signaling networks. PKCα is composed of modular domains that interact with each other to dynamically regulate spatial-temporal function. We find that PKCα specifically, rapidly and reversibly self-assembles in the presence of calcium in vitro.

Correlation between Activity and Domain Complementation in Adenylyl Cyclase Demonstrated with a Novel Fluorescence Resonance Energy Transfer Sensor.

Adenylyl cyclase (AC) activity relies on multiple effectors acting through distinct binding sites. Crystal structures have revealed the location of these sites, and biochemical studies have explored the kinetics of ACs, but the interplay between conformation and activity remains incompletely understood. Here, we describe a novel fluorescence resonance energy transfer (FRET) sensor that functions both as a soluble cyclase and a reporter of complementation within the catalytic domain.

Optofluidic lasers with a single molecular layer of gain.

We achieve optofluidic lasers with a single molecular layer of gain, in which green fluorescent protein, dye-labeled bovine serum albumin, and dye-labeled DNA, are used as the gain medium and attached to the surface of a ring resonator via surface immobilization biochemical methods. It is estimated that the surface density of the gain molecules is on the order of 10(12) cm(-2), sufficient for lasing under pulsed optical excitation. It is further shown that the optofluidic laser can be tuned by energy transfer mechanisms through biomolecular interactions.

Natural killer cells eradicate galectin-1-deficient glioma in the absence of adaptive immunity.

Natural killer (NK) cells safeguard against early tumor formation by destroying transformed target cells in a process referred to as NK immune surveillance. However, the immune escape mechanisms used by malignant brain tumors to subvert this innate type of immune surveillance remain unclear. Here we show that malignant glioma cells suppress NK immune surveillance by overexpressing the β-galactoside-binding lectin galectin-1.

Conserved modular domains team up to latch-open active protein kinase Cα.

Signaling proteins comprised of modular domains have evolved along with multicellularity as a method to facilitate increasing intracellular bandwidth. The effects of intramolecular interactions between modular domains within the context of native proteins have been largely unexplored. Here we examine intra- and intermolecular interactions in the multidomain signaling protein, protein kinase Cα (PKCα).

[A man with acute, painless swelling of the hand].

A 48-year-old man visited the hand surgery outpatient clinic with acute, painless swelling of the dorsal side of the right hand and fingers. We found erythema and crepitations of the skin. Laboratory tests showed no signs of infection.

Detection of G protein-selective G protein-coupled receptor (GPCR) conformations in live cells.

Although several recent studies have reported that GPCRs adopt multiple conformations, it remains unclear how subtle conformational changes are translated into divergent downstream responses. In this study, we report on a novel class of FRET-based sensors that can detect the ligand/mutagenic stabilization of GPCR conformations that promote interactions with G proteins in live cells. These sensors rely on the well characterized interaction between a GPCR and the C terminus of a Gα subunit.

Highly sensitive fluorescent protein FRET detection using optofluidic lasers.

We achieved optofluidic protein lasing using genetically encoded fluorescent protein FRET pairs linked by length-tunable peptides. Up to 25-fold reduction in the donor laser emission was observed when the donor and the acceptor were brought to close proximity, as compared to only 17% reduction in the donor emission using the conventional FRET detection. Our work opens a door to a broad range of applications in studying protein-protein interactions and protein-drug interactions.

Visualizing and manipulating focal adhesion kinase regulation in live cells.

Focal Adhesion Kinase (FAK) is essential for cell migration and plays an important role in tumor metastasis. However, the complex intermolecular and intramolecular interactions that regulate FAK activity at the focal adhesion remain unresolved. We have engineered a toolbox of FRET sensors that retain all of the individual FAK domains but modulate a key intramolecular regulatory interaction between the band 4.