Symptom Burden and Time from Symptom Onset to Cancer Diagnosis in Patients with Early-Onset Colorectal Cancer: A Multicenter Retrospective Analysis.

Background: The incidence of colorectal cancer (CRC) is decreasing in individuals >50 years due to organised screening but has increased for younger individuals. We characterized symptoms and their timing before diagnosis in young individuals. Methods: We identified all patients diagnosed with CRC between 1990-2017 in British Columbia, Canada. Individuals <50 years (n = 2544, EoCRC) and a matched cohort >50 (n = 2570, LoCRC) underwent chart review to identify CRC related symptoms at diagnosis and determine time from symptom onset to diagnosis. Results: Across all stages of CRC, EoCRC presented with significantly more symptoms than LoCRC (Stage 1 mean ± SD: 1.3 ± 0.9 vs. 0.7 ± 0.9, p = 0.0008; Stage 4: 3.3 ± 1.5 vs. 2.3 ± 1.7, p < 0.0001). Greater symptom burden at diagnosis was associated with worse survival in both EoCRC (p < 0.0001) and LoCRC (p < 0.0001). When controlling for cancer stage, both age (HR 0.87, 95% CI 0.8-1.0, p = 0.008) and increasing symptom number were independently associated with worse survival in multivariate models. Conclusions: Patients with EoCRC present with a greater number of symptoms of longer duration than LoCRC; however, time from patient reported symptom onset was not associated with worse outcomes.

Unmasking subtype-dependent susceptibility to C-type inactivation in mammalian Kv1 channels.

Shaker potassium channels have been an essential model for studying inactivation of ion channels and shaped our earliest understanding of N-type vs. C-type mechanisms. In early work describing C-type inactivation, López-Barneo and colleagues systematically characterized numerous mutations of Shaker residue T449, demonstrating that this position was a key determinant of C-type inactivation rate. In most of the closely related mammalian Kv1 channels, however, a persistent enigma has been that residue identity at this position has relatively modest effects on the rate of inactivation in response to long depolarizations. In this study, we report alternative ways to measure or elicit conformational changes in the outer pore associated with C-type inactivation. Using a strategically substituted cysteine in the outer pore, we demonstrate that mutation of Kv1.2 V381 (equivalent to Shaker T449) or W366 (Shaker W434) markedly increases susceptibility to modification by extracellularly applied MTSET. Moreover, due to the cooperative nature of C-type inactivation, Kv1.2 assembly in heteromeric channels markedly inhibits MTSET modification of this substituted cysteine in neighboring subunits. The identity of Kv1.2 residue V381 also markedly influences function in conditions that bias channels toward C-type inactivation, namely when Na+ is substituted for K+ as the permeant ion or when channels are blocked by an N-type inactivation particle (such as Kvß1.2). Overall, our findings illustrate that in mammalian Kv1 channels, the identity of the T449-equivalent residue can strongly influence function in certain experimental conditions, even while having modest effects on apparent inactivation during long depolarizations. These findings contribute to reconciling differences in experimental outcomes in many Kv1 channels vs. Shaker.

Control of Slc7a5 sensitivity by the voltage-sensing domain of Kv1 channels.

Many voltage-dependent ion channels are regulated by accessory proteins. We recently reported powerful regulation of Kv1.2 potassium channels by the amino acid transporter Slc7a5. In this study, we report that Kv1.1 channels are also regulated by Slc7a5, albeit with different functional outcomes. In heterologous expression systems, Kv1.1 exhibits prominent current enhancement ('disinhibition') with holding potentials more negative than -120 mV. Knockdown of endogenous Slc7a5 leads to larger Kv1.1 currents and strongly attenuates the disinhibition effect, suggesting that Slc7a5 regulation of Kv1.1 involves channel inhibition that can be reversed by supraphysiological hyperpolarizing voltages. We investigated chimeric combinations of Kv1.1 and Kv1.2, demonstrating that exchange of the voltage-sensing domain controls the sensitivity and response to Slc7a5, and localize a specific position in S1 with prominent effects on Slc7a5 sensitivity. Overall, our study highlights multiple Slc7a5-sensitive Kv1 subunits, and identifies the voltage-sensing domain as a determinant of Slc7a5 modulation of Kv1 channels.

Slc7a5 regulates Kv1.2 channels and modifies functional outcomes of epilepsy-linked channel mutations.

Kv1.2 is a prominent voltage-gated potassium channel that influences action potential generation and propagation in the central nervous system. We explored multi-protein complexes containing Kv1.2 using mass spectrometry followed by screening for effects on Kv1.2. We report that Slc7a5, a neutral amino acid transporter, has a profound impact on Kv1.2. Co-expression with Slc7a5 reduces total Kv1.2 protein, and dramatically hyperpolarizes the voltage-dependence of activation by -47 mV. These effects are attenuated by expression of Slc3a2, a known binding partner of Slc7a5. The profound Slc7a5-mediated current suppression is partly explained by a combination of gating effects including accelerated inactivation and a hyperpolarizing shift of channel activation, causing channels to accumulate in a non-conducting state. Two recently reported Slc7a5 mutations linked to neurodevelopmental delay exhibit a localization defect and have attenuated effects on Kv1.2. In addition, epilepsy-linked gain-of-function Kv1.2 mutants exhibit enhanced sensitivity to Slc7a5.

Extracellular redox sensitivity of Kv1.2 potassium channels.

Kv1.2 is a prominent potassium channel subtype in the nervous system and serves as an important structural template for investigation of ion channel function. However, Kv1.2 voltage-dependence exhibits dramatic cell-to-cell variability due to a gating mode shift that is regulated by an unknown mechanism. We report that this variable behavior is regulated by the extracellular redox environment. Exposure to reducing agents promotes a shift in gating properties towards an 'inhibited' gating mode that resists opening, and causes channels to exhibit pronounced use-dependent activation during trains of repetitive depolarizations. This sensitivity to extracellular redox potential is absent in other Kv1 channels, but is apparent in heteromeric channels containing Kv1.2 subunits, and overlaps with the reported physiological range of extracellular redox couples. Mutagenesis of candidate cysteine residues fails to abolish redox sensitivity. Therefore, we suggest that an extrinsic, redox-sensitive binding partner imparts these properties.

Oral administration of the nitroxide radical TEMPOL exhibits immunomodulatory and therapeutic properties in multiple sclerosis models.

Therapies with both immunomodulatory and neuroprotective properties are thought to have the greatest promise in reducing the severity and progression of multiple sclerosis (MS). Several reactive oxygen (ROS) and reactive nitrogen species (RNS) are implicated in inflammatory-mediated damage to the central nervous system (CNS) in MS and its animal model, experimental autoimmune encephalomyelitis (EAE). TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) is a stable nitroxide radical with potent antioxidant activity. The goal of our studies was to investigate the immunomodulatory effects and therapeutic potential of orally-delivered TEMPOL in the mouse EAE model. Mice receiving TEMPOL chow ad libitum for 2weeks prior to induction of active EAE showed delayed onset and reduced incidence of disease compared to control-fed animals. Reduced disease severity was associated with limited microglial activation and fewer inflammatory infiltrates. TEMPOL's effects were immunomodulatory, not immunosuppressive: T cells produced less interferon-γ and tumor necrosis factor-α, and TEMPOL-fed mice exhibited a shift towards TH2-type antibody responses. Both myeloid and myeloid-dendritic cells of TEMPOL-fed EAE animals had significantly lower levels of MHC class II expression than controls; CD40 was also significantly reduced. TEMPOL administration was associated with an enrichment of CD8+ T cell populations and CD4+FoxP3+ regulatory populations. TEMPOL reduced the severity of clinical disease when administered after the induction of disease, and also after the onset of clinical symptoms. To exclude effects on T cell priming in vivo, TEMPOL was tested with the passive transfer of encephalitogenic T cells and was found to reduce the incidence and peak severity of disease. Protection was associated with reduced infiltrates and a relative sparing of neurofilaments and axons. The ability of oral TEMPOL to reduce inflammation and axonal damage and loss demonstrate both anti-inflammatory and protective properties, with significant promise for the treatment of MS and related neurological disorders.

Cyclic Purine and Pyrimidine Nucleotides Bind to the HCN2 Ion Channel and Variably Promote C-Terminal Domain Interactions and Opening.

Cyclic AMP is thought to facilitate the opening of the HCN2 channel by binding to a C-terminal domain and promoting or inhibiting interactions between subunits. Here, we correlated the ability of cyclic nucleotides to promote interactions of isolated HCN2 C-terminal domains in solution with their ability to facilitate channel opening. Cyclic IMP, a cyclic purine nucleotide, and cCMP, a cyclic pyrimidine nucleotide, bind to a C-terminal domain containing the cyclic nucleotide-binding domain but, in contrast to other cyclic nucleotides examined, fail to promote its oligomerization, and produce only modest facilitation of opening of the full-length channel. Comparisons between ligand bound structures identify a region between the sixth and seventh ß strands and the distal C helix as important for facilitation and tight binding. We propose that promotion of interactions between the C-terminal domains by a given ligand contribute to its ability to facilitate opening of the full-length channel.

Determinants of frequency-dependent regulation of Kv1.2-containing potassium channels.

Voltage-gated potassium channels are important regulators of electrical excitation in many tissues, with Kv1.2 standing out as an essential contributor in the CNS. Genetic deletion of Kv1.2 invariably leads to early lethality in mice. In humans, mutations affecting Kv1.2 function are linked to epileptic encephalopathy and movement disorders. We have demonstrated that Kv1.2 is subject to a unique regulatory mechanism in which repetitive stimulation leads to dramatic potentiation of current. In this study, we explore the properties and molecular determinants of this use-dependent potentiation/activation. First, we examine how alterations in duty cycle (depolarization and repolarization/recovery times) affect the onset and extent of use-dependent activation. Also, we use trains of repetitive depolarizations to test the effects of a variety of Thr252 (S2-S3 linker) mutations on use-dependent activation. Substitutions of Thr with some sterically similar amino acids (Ser, Val, and Met, but not Cys) retain use-dependent activation, while bulky or charged amino acid substitutions eliminate use-dependence. Introduction of Thr at the equivalent position in other Kv1 channels (1.1, 1.3, 1.4), was not sufficient to transfer the phenotype. We hypothesize that use-dependent activation of Kv1.2 channels is mediated by an extrinsic regulator that binds preferentially to the channel closed state, with Thr252 being necessary but not sufficient for this interaction to alter channel function. These findings extend the conclusions of our recent demonstration of use-dependent activation of Kv1.2-containing channels in hippocampal neurons, by adding new details about the molecular mechanism underlying this effect.

Use-dependent activation of neuronal Kv1.2 channel complexes.

In excitable cells, ion channels are frequently challenged by repetitive stimuli, and their responses shape cellular behavior by regulating the duration and termination of bursts of action potentials. We have investigated the behavior of Shaker family voltage-gated potassium (Kv) channels subjected to repetitive stimuli, with a particular focus on Kv1.2. Genetic deletion of this subunit results in complete mortality within 2 weeks of birth in mice, highlighting a critical physiological role for Kv1.2. Kv1.2 channels exhibit a unique property described previously as "prepulse potentiation," in which activation by a depolarizing step facilitates activation in a subsequent pulse. In this study, we demonstrate that this property enables Kv1.2 channels to exhibit use-dependent activation during trains of very brief depolarizations. Also, Kv subunits usually assemble into heteromeric channels in the central nervous system, generating diversity of function and sensitivity to signaling mechanisms. We demonstrate that other Kv1 channel types do not exhibit use-dependent activation, but this property is conferred in heteromeric channel complexes containing even a single Kv1.2 subunit. This regulatory mechanism is observed in mammalian cell lines as well as primary cultures of hippocampal neurons. Our findings illustrate that use-dependent activation is a unique property of Kv1.2 that persists in heteromeric channel complexes and may influence function of hippocampal neurons.

Inward rectifiers and their regulation by endogenous polyamines.

Inwardly-rectifying potassium (Kir) channels contribute to maintenance of the resting membrane potential and regulation of electrical excitation in many cell types. Strongly rectifying Kir channels exhibit a very steep voltage dependence resulting in silencing of their activity at depolarized membrane voltages. The mechanism underlying this steep voltage dependence is blockade by endogenous polyamines. These small multifunctional, polyvalent metabolites enter the long Kir channel pore from the intracellular side, displacing multiple occupant ions as they migrate to a stable binding site in the transmembrane region of the channel. Numerous structure-function studies have revealed structural elements of Kir channels that determine their susceptibility to polyamine block, and enable the steep voltage dependence of this process. In addition, various channelopathies have been described that result from alteration of the polyamine sensitivity or activity of strongly rectifying channels. The primary focus of this article is to summarize current knowledge of the molecular mechanisms of polyamine block, and provide some perspective on lingering uncertainties related to this physiologically important mechanism of ion channel blockade. We also briefly review some of the important and well understood physiological roles of polyamine sensitive, strongly rectifying Kir channels, primarily of the Kir2 family.

In vivo emergence of a novel mutant L159F/L320F in the NS5B polymerase confers low-level resistance to the HCV polymerase inhibitors mericitabine and sofosbuvir.

BACKGROUND: Resistance to mericitabine (prodrug of HCV NS5B polymerase inhibitor PSI-6130) is rare and conferred by the NS5B S282T mutation. METHODS: Serum HCV RNA from patients who experienced viral breakthrough, partial response, or nonresponse in 2 clinical trials in which patients received mericitabine plus peginterferon alfa-2a (40KD)/ribavirin were analyzed by population and clonal sequence analysis as well as phenotypic assay for assessment of in vivo mericitabine resistance. RESULTS: Among 405 patients treated with mericitabine plus peginterferon alfa-2a/ribavirin in PROPEL and JUMP-C, virologic breakthrough or nonresponse were not observed; 12 patients experienced a partial response. The NS5B S282T resistance mutation was not observed in any patient. A number of treatment-associated NS5B changes were observed and characterized. A novel double mutant (L159F/L320F) with impaired replication capacity was detected in one HCV genotype 1b-infected patient. Introduction of double mutant L159F/L320F into genotype 1a (H77) and 1b (Con-1) replicons, respectively, increased the EC50 for mericitabine by 3.1- and 5.5-fold and the EC90 by 3.1- and 8.9-fold. The double mutant also decreased susceptibility to sofosbuvir (GS-7977) and GS-938 but not setrobuvir, relative to wild-type. CONCLUSIONS: A novel and replication-deficient double mutation (L159F/L320F) confers low-level resistance to mericitabine and cross-resistance to both sofosbuvir and GS-938. CLINICAL TRIALS REGISTRATION: NCT00869661, NCT01057667.