The bile acid-sensitive ion channel (BASIC) mediates bile acid-dependent currents in bile duct epithelial cells

The bile acid-sensitive ion channel (BASIC) is a member of the Deg/ENaC family of ion channels that is activated by bile acids. Despite the identification of cholangiocytes in the liver and unipolar brush cells in the cerebellum as sites of expression, the physiological function of BASIC in these cell types is not yet understood. Here we used a cholangiocyte cell line, normal rat cholangiocytes (NRCs), which expresses BASIC to study the role of the channel in epithelial transport using Ussing chamber experiments. Apical application of bile acids induced robust and transient increases in transepithelial currents that were carried by Na+ and partly blocked by the BASIC inhibitor diminazene. Genetic ablation of the BASIC gene in NRC using a CRISPR-cas9 approach resulted in a decrease of the bile acid-mediated response that matched the diminazene-sensitive current in NRC WT cells, suggesting that cholangiocytes respond to bile acids with a BASIC-mediated Na+ influx. Taken together, we have identified BASIC as a component of the cholangiocyte transport machinery, which might mediate a bile acid-dependent modification of the bile and thus control bile flux and composition.

The hypervariable region of atlastin-1 is a site for intrinsic and extrinsic regulation

Atlastin (ATL) GTPases catalyze homotypic membrane fusion of the peripheral endoplasmic reticulum (ER). GTP-hydrolysis–driven conformational changes and membrane tethering are prerequisites for proper membrane fusion. However, the molecular basis for regulation of these processes is poorly understood. Here we establish intrinsic and extrinsic modes of ATL1 regulation that involve the N-terminal hypervariable region (HVR) of ATLs. Crystal structures of ATL1 and ATL3 exhibit the HVR as a distinct, isoform-specific structural feature. Characterizing the functional role of ATL1’s HVR uncovered its positive effect on membrane tethering and on ATL1’s cellular function. The HVR is post-translationally regulated through phosphorylation-dependent modification. A kinase screen identified candidates that modify the HVR site specifically, corresponding to the modifications on ATL1 detected in cells. This work reveals how the HVR contributes to efficient and potentially regulated activity of ATLs, laying the foundation for the identification of cellular effectors of ATL-mediated membrane processes.

Spinal reconsolidation engages non-ionotropic NMDA receptor signaling to reverse pain hypersensitivity

Reconsolidation enables the activity-dependent modification of memory traces and has been used to reverse addiction, fear memory, and pain hypersensitivity in animal models. We demonstrate that non-ionotropic NMDA receptor signalling in the spinal dorsal horn is sufficient to reverse pain hypersensitivity and necessary for pain modulation by spinal reconsolidation. These findings reveal a key process by which reconsolidation weakens memory traces that may be exploited in the treatment of pain and other disorders.

Novel modification by L/F-tRNA-protein transferase (LFTR) generates a Leu/N-degron ligand in Escherichia coli.

The N-degron pathways are a set of proteolytic systems that relate the half-life of a protein to its N-terminal (Nt) residue. In Escherchia coli the principal N-degron pathway is known as the Leu/N-degron pathway of which an Nt Leu is a key feature of the degron. Although the physiological role of the Leu/N-degron pathway is currently unclear, many of the components of the pathway are well defined. Proteins degraded by this pathway contain an Nt degradation signal (N-degron) composed of an Nt primary destabilizing (Nd1) residue (Leu, Phe, Trp or Tyr) and an unstructured region which generally contains a hydrophobic element. Most N-degrons are generated from a pro-N-degron, either by endoproteolytic cleavage, or by enzymatic attachment of a Nd1 residue (Leu or Phe) to the N-terminus of a protein (or protein fragment) by the enzyme Leu/Phe tRNA protein transferase (LFTR) in a non-ribosomal manner. Regardless of the mode of generation, all Leu/N-degrons are recognized by ClpS and delivered to the ClpAP protease for degradation. To date, only two physiological Leu/N-degron bearing substrates have been verified, one of which (PATase) is modified by LFTR. In this study, we have examined the substrate proteome of LFTR during stationary phase. From this analysis, we have identified several additional physiological Leu/N-degron ligands, including AldB, which is modified by a previously undescribed activity of LFTR. Importantly, the novel specificity of LFTR was confirmed in vitro, using a range of model proteins. Our data shows that processing of the Nt-Met of AldB generates a novel substrate for LFTR. Importantly, the LFTR-dependent modification of T2-AldB is essential for its turnover by ClpAPS, in vitro. To further examine the acceptor specificity of LFTR, we performed a systematic analysis using a series of peptide arrays. These data reveal that the identity of the second residue modulates substrate conjugation with positively charged residues being favored and negatively charged and aromatic residues being disfavored. Collectively, these findings extend our understanding of LFTR specificity and the Leu/N-degron pathway in E. coli.

Methylation of viral mRNA cap structures by PCIF1 attenuates the antiviral activity of interferon-β

Interferons induce cell-intrinsic responses associated with resistance to viral infection. To overcome the suppressive action of interferons and their effectors, viruses have evolved diverse mechanisms. Using vesicular stomatitis virus (VSV), we report that the host cell N6-adenosine messenger RNA (mRNA) cap methylase, phosphorylated C-terminal domain interacting factor 1 (PCIF1), attenuates the antiviral response. We employed cell-based and in vitro biochemical assays to demonstrate that PCIF1 efficiently modifies VSV mRNA cap structures to m7Gpppm6Am and define the substrate requirements for this modification. Functional assays revealed that the PCIF1-dependent modification of VSV mRNA cap structures is inert with regard to mRNA stability, translation, and viral infectivity but attenuates the antiviral effects of the treatment of cells with interferon-β. Cells lacking PCIF1 or expressing a catalytically inactive PCIF1 exhibit an augmented inhibition of viral replication and gene expression following interferon-β treatment. We further demonstrate that the mRNA cap structures of rabies and measles viruses are also modified by PCIF1 to m7Gpppm6Am. This work identifies a function of PCIF1 and cap-proximal m6Am in attenuation of the host response to VSV infection that likely extends to other viruses.

Serine-linked PARP1 auto-modification controls PARP inhibitor response

Poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 are recruited and activated by DNA damage, resulting in ADP-ribosylation at numerous sites, both within PARP1 itself and in other proteins. Several PARP1 and PARP2 inhibitors are currently employed in the clinic or undergoing trials for treatment of various cancers. These drugs act primarily by trapping PARP1 on damaged chromatin, which can lead to cell death, especially in cells with DNA repair defects. Although PARP1 trapping is thought to be caused primarily by the catalytic inhibition of PARP-dependent modification, implying that ADP-ribosylation (ADPr) can counteract trapping, it is not known which exact sites are important for this process. Following recent findings that PARP1- or PARP2-mediated modification is predominantly serine-linked, we demonstrate here that serine ADPr plays a vital role in cellular responses to PARP1/PARP2 inhibitors. Specifically, we identify three serine residues within PARP1 (499, 507, and 519) as key sites whose efficient HPF1-dependent modification counters PARP1 trapping and contributes to inhibitor tolerance. Our data implicate genes that encode serine-specific ADPr regulators, HPF1 and ARH3, as potential PARP1/PARP2 inhibitor therapy biomarkers.

Calcineurin Participation in Hebbian and Homeostatic Plasticity Associated With Extinction

In nature, animals need to adapt to constant changes in their environment. Learning and memory are cognitive capabilities that allow this to happen. Extinction, the reduction of a certain behavior or learning previously established, refers to a very particular and interesting type of learning that has been the basis of a series of therapies to diminish non-adaptive behaviors. In recent years, the exploration of the cellular and molecular mechanisms underlying this type of learning has received increasing attention. Hebbian plasticity (the activity-dependent modification of the strength or efficacy of synaptic transmission), and homeostatic plasticity (the homeostatic regulation of plasticity) constitute processes intimately associated with memory formation and maintenance. Particularly, long-term depression (LTD) has been proposed as the underlying mechanism of extinction, while the protein phosphatase calcineurin (CaN) has been widely related to both the extinction process and LTD. In this review, we focus on the available evidence that sustains CaN modulation of LTD and its association with extinction. Beyond the classic view, we also examine the interconnection among extinction, Hebbian and homeostatic plasticity, as well as emergent evidence of the participation of kinases and long-term potentiation (LTP) on extinction learning, highlighting the importance of the balance between kinases and phosphatases in the expression of extinction. Finally, we also integrate data that shows the association between extinction and less-studied phenomena, such as synaptic silencing and engram formation that open new perspectives in the field.

After-hyperpolarization promotes the firing of mitral cells through a voltage dependent modification of action potential threshold

In the olfactory bulb (OB), mitral cells (MCs) display a spontaneous firing that is characterized by bursts of action potentials intermixed with silent periods. Burst firing frequency and duration are heterogeneous among MCs and increase with membrane depolarization. By using patch clamp recording on rat slices, we dissected out the intrinsic properties responsible of this activity. We showed that the threshold of action potential (AP) generation dynamically changes as a function of the trajectory of the membrane potential; becoming more negative when the membrane was hyperpolarized and having a recovering rate, inversely proportional to the membrane repolarization rate. Such variations appeared to be produced by changes in the inactivation state of voltage dependent Na+ channels. Thus, the modification AP threshold favours the initiation of the burst following hyperpolarizing event such as negative membrane oscillations or inhibitory transmission. After the first AP, the following afterhyperpolarization (AHP) brought the threshold just below the membrane resting potential or within membrane oscillations and, as a consequence, the threshold was exceeded during the fast repolarization component of the AHP. In this way the fast AHP acts as a regenerative mechanism that sustains the firing. Bursts were stopped by the development of a slow repolarization component of the AHP. The AHP characteristics appeared as determining the bursting properties; AHP with larger amplitudes and faster repolarizations being associated with longer and higher frequency bursts. Thus, the increase of bursts length and frequency upon membrane depolarization would be attributable to the modifications of the AHP and of Na+ channels inactivation.

Confinement effects for rubberised concrete in tubular steel cross-sections under combined loading

This paper describes an experimental investigation into confinement effects provided by circular tubular sections to rubberised concrete materials under combined loading. The tests include specimens with 0%, 30% and 60% rubber replacement of mineral aggregates by volume. After describing the experimental arrangements and specimen details, the results of bending and eccentric compression tests are presented, together with complementary axial compression tests on stub-column samples. Tests on hollow steel specimens are also included for comparison purposes. Particular focus is given to assessing the confinement effects in the infill concrete as well as their influence on the axial–bending cross-section strength interaction. The results show that whilst the capacity is reduced with the increase in the rubber replacement ratio, an enhanced confinement action is obtained for high rubber content concrete compared with conventional materials. Test measurements by means of digital image correlation techniques show that the confinement in axial compression and the neutral axis position under combined loading depend on the rubber content. Analytical procedures for determining the capacity of rubberised concrete infilled cross-sections are also considered based on the test results as well as those from a collated database and then compared with available recommendations. Rubber content-dependent modification factors are proposed to provide more realistic representations of the axial and flexural cross-section capacities. The test results and observations are used, in conjunction with a number of analytical assessments, to highlight the main parameters influencing the behaviour and to propose simplified expressions for determining the cross-section strength under combined compression and bending.

Electrolyte-Dependent Modification of Resistive Switching in Anodic Hafnia

Anodic HfO2 memristors grown in phosphate, borate, or citrate electrolytes and formed on sputtered Hf with Pt top electrodes are characterized at fundamental and device levels. The incorporation of electrolyte species deep into anodic memristors concomitant with HfO2 crystalline structure conservation is demonstrated by elemental analysis and atomic scale imaging. Upon electroforming, retention and endurance tests are performed on memristors. The use of borate results in the weakest memristive performance while the citrate demonstrates clear superior memristive properties with multilevel switching capabilities and high read/write cycling in the range of 106. Low temperature heating applied to memristors shows a direct influence on their behavior mainly due to surface release of water. Citrate-based memristors show remarkable properties independent on device operation temperatures up to 100 °C. The switching dynamic of anodic HfO2 memristors is discussed by analyzing high resolution transmission electron microscope images. Full and partial conductive filaments are visualized, and apart from their modeling, a concurrency of filaments is additionally observed. This is responsible for the multilevel switching mechanism in HfO2 and is related to device failure mechanisms.