Cloning and expression of a zebrafish SCN1B ortholog and identification of a species-specific splice variant

Abstract Background Voltage-gated Na+ channel β1 (Scn1b) subunits are multi-functional proteins that play roles in current modulation, channel cell surface expression, cell adhesion, cell migration, and neurite outgrowth. We have shown previously that β1 modulates electrical excitability in vivo using a mouse model. Scn1b null mice exhibit spontaneous seizures and ataxia, slowed action potential conduction, decreased numbers of nodes of Ranvier in myelinated axons, alterations in nodal architecture, and differences in Na+ channel α subunit localization. The early death of these mice at postnatal day 19, however, make them a challenging model system to study. As a first step toward development of an alternative model to investigate the physiological roles of β1 subunits in vivo we cloned two β1-like subunit cDNAs from D. rerio. Results Two β1-like subunit mRNAs from zebrafish, scn1ba_tv1 and scn1ba_tv2, arise from alternative splicing of scn1ba. The deduced amino acid sequences of Scn1ba_tv1 and Scn1ba_tv2 are identical except for their C-terminal domains. The C-terminus of Scn1ba_tv1 contains a tyrosine residue similar to that found to be critical for ankyrin association and Na+ channel modulation in mammalian β1. In contrast, Scn1ba_tv2 contains a unique, species-specific C-terminal domain that does not contain a tyrosine. Immunohistochemical analysis shows that, while the expression patterns of Scn1ba_tv1 and Scn1ba_tv2 overlap in some areas of the brain, retina, spinal cord, and skeletal muscle, only Scn1ba_tv1 is expressed in optic nerve where its staining pattern suggests nodal expression. Both scn1ba splice forms modulate Na+ currents expressed by zebrafish scn8aa, resulting in shifts in channel gating mode, increased current amplitude, negative shifts in the voltage dependence of current activation and inactivation, and increases in the rate of recovery from inactivation, similar to the function of mammalian β1 subunits. In contrast to mammalian β1, however, neither zebrafish subunit produces a complete shift to the fast gating mode and neither subunit produces complete channel inactivation or recovery from inactivation. Conclusion These data add to our understanding of structure-function relationships in Na+ channel β1 subunits and establish zebrafish as an ideal system in which to determine the contribution of scn1ba to electrical excitability in vivo.http://deepblue.lib.umich.edu/bitstream/2027.42/112585/1/12864_2007_Article_939.pd

Efficacy and safety of baricitinib or ravulizumab in adult patients with severe COVID-19 (TACTIC-R): a randomised, parallel-arm, open-label, phase 4 trial

Background From early in the COVID-19 pandemic, evidence suggested a role for cytokine dysregulation and complement activation in severe disease. In the TACTIC-R trial, we evaluated the efficacy and safety of baricitinib, an inhibitor of Janus kinase 1 (JAK1) and JAK2, and ravulizumab, a monoclonal inhibitor of complement C5 activation, as an adjunct to standard of care for the treatment of adult patients hospitalised with COVID-19. Methods TACTIC-R was a phase 4, randomised, parallel-arm, open-label platform trial that was undertaken in the UK with urgent public health designation to assess the potential of repurposing immunosuppressants for the treatment of severe COVID-19, stratified by a risk score. Adult participants (aged ≥18 years) were enrolled from 22 hospitals across the UK. Patients with a risk score indicating a 40% risk of admission to an intensive care unit or death were randomly assigned 1:1:1 to standard of care alone, standard of care with baricitinib, or standard of care with ravulizumab. The composite primary outcome was the time from randomisation to incidence (up to and including day 14) of the first event of death, invasive mechanical ventilation, extracorporeal membrane oxygenation, cardiovascular organ support, or renal failure. The primary interim analysis was triggered when 125 patient datasets were available up to day 14 in each study group and we included in the analysis all participants who were randomly assigned. The trial was registered on ClinicalTrials.gov (NCT04390464). Findings Between May 8, 2020, and May 7, 2021, 417 participants were recruited and randomly assigned to standard of care alone (145 patients), baricitinib (137 patients), or ravulizumab (135 patients). Only 54 (39%) of 137 patients in the baricitinib group received the maximum 14-day course, whereas 132 (98%) of 135 patients in the ravulizumab group received the intended dose. The trial was stopped after the primary interim analysis on grounds of futility. The estimated hazard ratio (HR) for reaching the composite primary endpoint was 1·11 (95% CI 0·62–1·99) for patients on baricitinib compared with standard of care alone, and 1·53 (0·88–2·67) for ravulizumab compared with standard of care alone. 45 serious adverse events (21 deaths) were reported in the standard-of-care group, 57 (24 deaths) in the baricitinib group, and 60 (18 deaths) in the ravulizumab group. Interpretation Neither baricitinib nor ravulizumab, as administered in this study, was effective in reducing disease severity in patients selected for severe COVID-19. Safety was similar between treatments and standard of care. The short period of dosing with baricitinib might explain the discrepancy between our findings and those of other trials. The therapeutic potential of targeting complement C5 activation product C5a, rather than the cleavage of C5, warrants further evaluation

Effect of remote ischaemic conditioning on clinical outcomes in patients with acute myocardial infarction (CONDI-2/ERIC-PPCI): a single-blind randomised controlled trial.

BACKGROUND: Remote ischaemic conditioning with transient ischaemia and reperfusion applied to the arm has been shown to reduce myocardial infarct size in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). We investigated whether remote ischaemic conditioning could reduce the incidence of cardiac death and hospitalisation for heart failure at 12 months. METHODS: We did an international investigator-initiated, prospective, single-blind, randomised controlled trial (CONDI-2/ERIC-PPCI) at 33 centres across the UK, Denmark, Spain, and Serbia. Patients (age >18 years) with suspected STEMI and who were eligible for PPCI were randomly allocated (1:1, stratified by centre with a permuted block method) to receive standard treatment (including a sham simulated remote ischaemic conditioning intervention at UK sites only) or remote ischaemic conditioning treatment (intermittent ischaemia and reperfusion applied to the arm through four cycles of 5-min inflation and 5-min deflation of an automated cuff device) before PPCI. Investigators responsible for data collection and outcome assessment were masked to treatment allocation. The primary combined endpoint was cardiac death or hospitalisation for heart failure at 12 months in the intention-to-treat population. This trial is registered with ClinicalTrials.gov (NCT02342522) and is completed. FINDINGS: Between Nov 6, 2013, and March 31, 2018, 5401 patients were randomly allocated to either the control group (n=2701) or the remote ischaemic conditioning group (n=2700). After exclusion of patients upon hospital arrival or loss to follow-up, 2569 patients in the control group and 2546 in the intervention group were included in the intention-to-treat analysis. At 12 months post-PPCI, the Kaplan-Meier-estimated frequencies of cardiac death or hospitalisation for heart failure (the primary endpoint) were 220 (8·6%) patients in the control group and 239 (9·4%) in the remote ischaemic conditioning group (hazard ratio 1·10 [95% CI 0·91-1·32], p=0·32 for intervention versus control). No important unexpected adverse events or side effects of remote ischaemic conditioning were observed. INTERPRETATION: Remote ischaemic conditioning does not improve clinical outcomes (cardiac death or hospitalisation for heart failure) at 12 months in patients with STEMI undergoing PPCI. FUNDING: British Heart Foundation, University College London Hospitals/University College London Biomedical Research Centre, Danish Innovation Foundation, Novo Nordisk Foundation, TrygFonden

Adding 6 months of androgen deprivation therapy to postoperative radiotherapy for prostate cancer: a comparison of short-course versus no androgen deprivation therapy in the RADICALS-HD randomised controlled trial

Background Previous evidence indicates that adjuvant, short-course androgen deprivation therapy (ADT) improves metastasis-free survival when given with primary radiotherapy for intermediate-risk and high-risk localised prostate cancer. However, the value of ADT with postoperative radiotherapy after radical prostatectomy is unclear. Methods RADICALS-HD was an international randomised controlled trial to test the efficacy of ADT used in combination with postoperative radiotherapy for prostate cancer. Key eligibility criteria were indication for radiotherapy after radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to radiotherapy alone (no ADT) or radiotherapy with 6 months of ADT (short-course ADT), using monthly subcutaneous gonadotropin-releasing hormone analogue injections, daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as distant metastasis arising from prostate cancer or death from any cause. Standard survival analysis methods were used, accounting for randomisation stratification factors. The trial had 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 80% to 86% (hazard ratio [HR] 0·67). Analyses followed the intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov, NCT00541047. Findings Between Nov 22, 2007, and June 29, 2015, 1480 patients (median age 66 years [IQR 61–69]) were randomly assigned to receive no ADT (n=737) or short-course ADT (n=743) in addition to postoperative radiotherapy at 121 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 9·0 years (IQR 7·1–10·1), metastasis-free survival events were reported for 268 participants (142 in the no ADT group and 126 in the short-course ADT group; HR 0·886 [95% CI 0·688–1·140], p=0·35). 10-year metastasis-free survival was 79·2% (95% CI 75·4–82·5) in the no ADT group and 80·4% (76·6–83·6) in the short-course ADT group. Toxicity of grade 3 or higher was reported for 121 (17%) of 737 participants in the no ADT group and 100 (14%) of 743 in the short-course ADT group (p=0·15), with no treatment-related deaths. Interpretation Metastatic disease is uncommon following postoperative bed radiotherapy after radical prostatectomy. Adding 6 months of ADT to this radiotherapy did not improve metastasis-free survival compared with no ADT. These findings do not support the use of short-course ADT with postoperative radiotherapy in this patient population

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

A molecular basis for the different local anesthetic affinities of resting versus open and inactivated states of the sodium channel. Mol Pharmacol 55:134–141

ABSTRACT Voltage-gated sodium channels are inhibited by local anesthetic drugs. This inhibition has complex voltage-and frequency-dependent properties, consistent with a model in which the sodium channel has low affinity for local anesthetics when it is in resting states and higher affinity when it is in open or inactivated states. Two residues, a phenylalanine (F1710) and a tyrosine (Y1717), in transmembrane segment IVS6 of the channel ␣ subunit are critical for state-dependent block. We examined how these residues determine channel sensitivity to local anesthetics by introducing mutations that varied their size, hydrophobicity, and aromaticity. Block of resting channels by tetracaine was correlated with hydrophobicity at position 1710, as if hydrophobic drug-receptor interactions stabilize binding to resting states. In contrast, drug action on open or inactivated channels required an aromatic residue at this position. We propose that the native phenylalanine at position 1710 stabilizes drug binding to open or inactivated states by either cation-or aromatic-aromatic interactions between the aromatic side chain of the amino acid and charged or aromatic moieties on the drug molecule. We also consider the alternative possibility that mutations at this position affect drug action by either altering access to the receptor or by allosteric changes in receptor conformation. Mutations at position 1717 also altered drug action; however, these effects were not well-correlated with the size, hydrophobicity, or aromaticity of the substituted amino acid. These results suggest that the residue at this position does not contribute directly to the drug receptor

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-7

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p>acetylated α-tubulin. : Anti Scn1ba_tv1 (green), anti-acetylated α-tubulin (red). : Anti-Scn1ba_tv2 (green), anti-acetylated α-tubulin (red). Images were viewed with an Olympus FluoView 500 confocal microscope at 100× magnification with 5× additional zoom. Scale bar: 50 μm

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-6

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p> α-tubulin (red). Anti-Scn1ba_tv2 stains the layers of the retina, including the ganglion cell layer (GCL), inner plexiform layer (IPL), outer plexiform layer (OPL), outer limiting membrane (OLM), and photoreceptor cell layer (PR). Staining appears to be absent in the inner nuclear layer (INL) and in the optic nerve (on). Anti-Scn1ba_tv1 stains all the layers of the retina including the inner nuclear layer, where it shows robust staining. In contrast to anti-Scn1ba_tv2, anti-Scn1ba_tv1 labels optic nerve. Scale bar: 50 μm

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-11

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p>that are identical are indicted in red, strongly similar substitutions are indicated by (:), and weakly similar amino acids are indicated by (.). Identical resides in exon 5 of Scn1ba_tv1 and Scn1b are indicated in green. The two cysteine residues predicted to form the Ig loop are indicated in blue. The conserved regions that form the A/A' face of the Ig loop, sites of interaction with theα subunit [17], are underlined. Tyrosine-181 in Scn1b and the corresponding residues in Scn1ba_tv1 and Scn1ba_tv2 are highlighted in yellow. Predicted sites of N-linked glycosylation are indicated by ▼. These sites were determined using NetNGlyc 1.0 [61]. Transmembrane segments are indicated as boxes. Peptides used for antibody generation are underlined in blue. Predicted β-sheets in the Ig loop domain, based on the crystal structure of myelin P[62], are shown with labeled arrows and correspond to the ribbon diagram included in the lower panel. Lower panel: Proposed three-dimensional structure of the Ig domain of β1 using the crystal structure of myelin Po (PDB 1NEU) as a template. The figure was created with the KiNG Viewer program via the RCSB Protein Data Bank web site [63]. β strands corresponding to the arrows in the upper panel are labeled A through G. . Schematic showing the genomic organization of zebrafish . The positions of introns 1 through 5 (I1 – I5) are indicated. Positions of primers used for RT-PCR in panel D are indicated. The C-terminal alternate splice domains contained in and are encoded by exon 5. . Model of alternative splicing of . Exons 4 and 5 (boxes) and intron 4 (line) are illustrated. The splice acceptor sequence at the beginning of exon 5 is indicated by and the internal alternate splice acceptor site in exon 5 is indicated by a dashed line and by ▼. The location of stop codons in the resulting mRNAs are indicated. Drawings are not to scale. Consensus splice acceptor sequence [22] and the acceptor sequences found in exon 5 are indicated in the lower portion of the panel. P: pyrimidine. P: purine. Lower case: intronic sequence. Upper case: exonic sequence. The "T" indicated by the red arrow in the internal, alternate acceptor is rare and significantly weakens the site [22]. . RT-PCR from whole fish RNA demonstrating that both splice variants of are expressed in the mRNA pool. The upper band corresponds to and the lower band corresponds to . Translations of the resulting alternate C-terminal splice products are shown below. The sequence highlighted in green is found in Scn1ba_tv1 and corresponds to the green portion of exon 5 illustrated in panel C. The sequence highlighted in turquoise is found in Scn1ba_tv2 and corresponds to the turquoise portion of exon 5 illustrated in panel C

Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant-4

<p><b>Copyright information:</b></p><p>Taken from "Cloning and expression of a zebrafish ortholog and identification of a species-specific splice variant"</p><p>http://www.biomedcentral.com/1471-2164/8/226</p><p>BMC Genomics 2007;8():226-226.</p><p>Published online 10 Jul 2007</p><p>PMCID:PMC1965480.</p><p></p> α-tubulin (red). OP: olfactory pit. Scale bar: 50 μm