Randomized Clinical Trial of Endovascular Therapy for Acute Large Vessel Occlusion with Large Ischemic Core (RESCUE-Japan LIMIT): Rationale and Study Protocol

Endovascular therapy is strongly recommended for acute cerebral large vessel occlusion (LVO) with an Alberta stroke program early computed tomography score (ASPECTS) ≥6 due to occlusion of the internal carotid artery or M1 segment of the middle cerebral artery. However, the effect of endovascular therapy for patients with a large ischemic core with an ASPECTS ≤5 (0–5) was not established. A multicenter, randomized, open-label, parallel-group trial was conducted to investigate the superiority of endovascular therapy over medical therapy without endovascular therapy for a large ischemic core with ASPECTS (3–5). Patients were randomly assigned to receive endovascular therapy or without endovascular therapy at a ratio of 1:1. The primary outcome was a moderate functional outcome, defined as a modified Rankin scale (mRS; scores ranging from 0 [no symptoms] to 6 [death]) ≤3 after 90 days. The secondary outcomes were defined as ordinal mRS, good functional outcome (mRS ≤2), excellent functional outcome (mRS ≤1), mRS shift analysis after 90 days, and early improvement of neurological findings at 48 hours. A total sample size of 200 was estimated to provide a power of 0.9 with a two-sided alpha of 0.05, for the primary outcome, considering a 15% dropout rate. This randomized clinical trial reported the applicability of endovascular therapy in patients with acute cerebral LVO with a large ischemic core.     

High Protein Diet Feeding Aggravates Hyperaminoacidemia in Mice Deficient in Proglucagon-Derived Peptides

(1) Background: Protein stimulates the secretion of glucagon (GCG), which can affect glucose metabolism. This study aimed to analyze the metabolic effect of a high-protein diet (HPD) in the presence or absence of proglucagon-derived peptides, including GCG and GLP-1. (2) Methods: The response to HPD feeding for 7 days was analyzed in mice deficient in proglucagon-derived peptides (GCGKO). (3) Results: In both control and GCGKO mice, food intake and body weight decreased with HPD and intestinal expression of Pepck increased. HPD also decreased plasma FGF21 levels, regardless of the presence of proglucagon-derived peptides. In control mice, HPD increased the hepatic expression of enzymes involved in amino acid metabolism without the elevation of plasma amino acid levels, except branched-chain amino acids. On the other hand, HPD-induced changes in the hepatic gene expression were attenuated in GCGKO mice, resulting in marked hyperaminoacidemia with lower blood glucose levels; the plasma concentration of glutamine exceeded that of glucose in HPD-fed GCGKO mice. (4) Conclusions: Increased plasma amino acid levels are a common feature in animal models with blocked GCG activity, and our results underscore that GCG plays essential roles in the homeostasis of amino acid metabolism in response to altered protein intake.     

Increased serum amiodarone concentration in hypertriglyceridemic patients: Effects of drug distribution to serum lipoproteins

Amiodarone and its main metabolite, desethylamiodarone (DEA), are highly distributed to serum lipoproteins such as very‐low‐density lipoprotein (VLDL) and low‐density lipoprotein (LDL), which are the carriers of triglyceride and cholesterol. This study aimed to investigate the association of serum concentrations of amiodarone and DEA with the levels of serum lipids in terms of drug distribution to lipoprotein fractions in patients with hyperlipidemia. Total serum concentrations of amiodarone and DEA were examined in 116 patients receiving amiodarone for tachyarrhythmias. The concentration‐to‐dose (C/D) ratio of amiodarone positively correlated with the level of serum triglyceride (r_s  = 0.541, p < 0.001) and was higher in the hypertriglyceridemic state than in normotriglyceridemic state (479 ± 211 vs. 320 ± 161, p < 0.001). No correlation was found between the C/D ratio of DEA and serum triglyceride levels (r_s  = 0.272), although higher values were observed in the hypertriglyceridemic state (322 ± 125 vs. 285 ± 143, p < 0.001). In the hypertriglyceridemic state, the distribution of amiodarone increased in LDL/VLDL fraction and decreased in high‐density lipoprotein and albumin fractions. The ratio of serum amiodarone to serum DEA, a metabolic ratio of amiodarone, positively correlated with serum triglyceride levels (r_s  = 0.572, p < 0.001) and was higher in the hypertriglyceridemic state, suggesting that amiodarone metabolism decreased in hyperlipidemia. The results of this study reveal that serum concentrations of amiodarone increase in the hypertriglyceridemic state through the increased lipoprotein‐binding and decreased metabolism of amiodarone.

Study Highlights

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

Lipoproteins can carry not only serum lipids but also certain lipophilic compounds such as amiodarone. Changes in the lipoprotein‐binding of amiodarone may lead to highly variable pharmacokinetics and poor concentration–effect relationships of the drug.

• WHAT QUESTION DID THIS STUDY ADDRESS?

This study addressed the association between serum amiodarone concentration and serum lipid levels in patients with arrhythmia, as well as the lipoprotein‐binding and metabolism of amiodarone in the hyperlipidemic state.

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

Serum amiodarone concentration is increased in patients with hypertriglyceridemia and it is positively correlated with serum triglyceride levels. These results are attributable to an increase in the circulating lipoprotein‐bound form of amiodarone and decreased metabolism of the drug in the hypertriglyceridemic state.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

Changes in the lipoprotein‐binding of amiodarone can help explain differences in the pharmacokinetics and pharmacodynamics of amiodarone related to normotriglyceridemic and hypertriglyceridemic states.     

Estimating pulmonary capillary wedge pressures using Doppler variables of early diastolic left ventricular inflow

The present study was performed to determine whether a multilineal regression model based on the early diastolic transmitral flow peak velocity (E) and the propagation velocity of early diastolic inflow (PV) could estimate the pulmonary capillary wedge pressure (PCWP). PCWP and Doppler variables were simultaneously recorded in 30 patients. PCWP was estimated by multilinear regression analysis using E and PV. The predictive accuracy of the equation obtained from the analysis was tested prospectively in a separate group of 65 patients divided into 3 groups: left ventricular (LV) systolic dysfunction (Group A), LV hypertrophy (Group B), and preserved systolic function without hypertrophy (Group C). The initial results obtained in groups B and C, respectively, were: r=0.77; r=0.81. These results indicate that a multilinear regression model based on E and PV is a noninvasive method of accurately estimating PCWP in a variety of cardiac disease states.     

Conformational alterations in unidirectional ion transport of a light-driven chloride pump revealed using X-ray free electron lasers

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10-µs and 1-ms time points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied by a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.

Microbial ion-pumping rhodopsins are integral membrane proteins that actively transport ions across membranes upon light stimulation (1). Bacteriorhodopsin (bR) and halorhodopsin (HR) are well-known microbial ion-pumping rhodopsins found in halophilic archaea (2, 3). bR is a light-driven outward proton pump and HR is a light-driven inward anion pump, specific for chloride ion. Microbial ion-pumping rhodopsins possess common structural features consisting of seven α-helices with an all-trans retinal covalently bound to a lysine residue as the chromophore, despite the transport of different ions (4). The retinal undergoes photoisomerization from the all-trans to 13-cis configuration, which initiates the photocycle accompanied by several intermediates to export ions (4, 5). Its light-controllable function is suitable for optogenetics applications for manipulating cells, such as neurons, by changing the ion concentration inside or outside the membrane (6, 7). In fact, microbial rhodopsins, including channelrhodopsins and HRs, are employed as optogenetic tools (8–10).Nonlabens marinus rhodopsin-3 (NM-R3) is a light-driven chloride pump recently discovered in a marine flavobacterium (11). It is a distinct chloride pump from HRs and shows low amino acid sequence homology with HRs (11). To date, HR-type chloride pumps have been found in haloarchaea, marine bacteria, and cyanobacteria, including Halobacterium salinarum, Natronomonas pharaonis, and Mastigocladopsins repens, with sequence identities of 20%, 21%, and 20% to NM-R3, respectively (3, 12–15). Interestingly, NM-R3 has higher sequence identity (36%) to Krokinobacter rhodopsin 2 (KR2), a sodium pump found in Krokinobacter eikastus (16). NM-R3 possesses a unique NTQ motif (Asn98, Thr102, Gln109) in the third helix (helix C), which corresponds to key residues (DTD motif, Asp85, Thr89, Asp96) for proton transport in bR (11, 17, 18) (SI Appendix, Table S1). Asp85 acts as the primary proton acceptor of bR from the protonated Schiff-base (PSB), with assistance from Thr89 and Asp96, which is the proton donor (5, 17, 18). HRs from haloarchaea have a highly conserved TSA (Thr, Ser, Ala) motif, while the Ala residue is replaced by Asp in HR from cyanobacteria (19). In the X-ray crystal structure of NM-R3 (SI Appendix, Fig. S1A), a chloride ion located between the PSB and Asn98 (SI Appendix, Fig. S1B) is stabilized by the positive charge of the PSB (20). The position of this chloride ion is similar to those in the H. salinarum HR and N. pharaonis HR (NpHR) structures except for Thr and Ser, which correspond to Asn98 and Thr102 in NM-R3, respectively (20–22). Several amino acid residues around the retinal, including Arg95, Trp99, Trp201, and Asp231, are highly conserved among ion-pumping rhodopsins. Previous spectroscopic studies suggested that NM-R3 displays a similar sequence of intermediates, with K-, L-, N-, and O-like species, as in other HRs (23) (Fig. 1A). Recently, intermediate structures of NM-R3 obtained by low-temperature trapping X-ray crystallography and serial femtosecond crystallography (SFX) have been reported (24, 25). However, the detailed ion-pump mechanism still remains unclear, due to the lack of dynamic structures of anion transport at atomic resolution.Open in a separate windowFig. 1.TR-visible absorption spectroscopy for microcrystals. (A) Photocycle model of NM-R3 in the 1 M NaCl buffer solution (23). (B) TR difference spectra ΔA upon the 532-nm excitation. The difference was calculated by subtracting the spectrum of NM-R3. (C) Global fitting analysis with two exponentials. The A₁ and A₂ amplitude spectra correspond to the differences of [ΔA_O – ΔA_{10 µs}] and [ΔA_{200 ms} − ΔA_O], respectively. Here, ΔA_O represents the difference spectrum of the O intermediate minus NM-R3. (D) The isomeric forms of the retinal chromophore in bacterial-type rhodopsins.Time-resolved serial femtosecond crystallography (TR-SFX) is a powerful tool for visualizing reactions and motions in proteins at the atomic level (26–28). In SFX, myriads of microcrystals are continuously injected by a sample injector into an irradiation point of X-ray free electron lasers (XFELs) at room temperature, thus providing diffraction patterns before the onset of radiation damage by the intense X-ray pulse. Combined with a visible-light pump laser for reaction initiation, TR-SFX has been applied to light-driven ion pumps to observe the structural dynamics during the ion transfer. While TR-SFX has revealed femto-to-millisecond structural dynamics in light-driven cation pumps, including bR and KR2 (29–31), TR-SFX studies of anion pumps have been limited to early-stage structures adopted at picoseconds after light illumination (32). In addition, although NM-R3 pumps a chloride ion (Cl⁻) as a physiological substrate, it can also transport bromide (Br⁻), iodide (I⁻), and other anions from the extracellular side to the cytoplasmic side (23). I⁻ or Br⁻ serves as a marker for tracking the positions of ions, due to the greater number of electrons, whereas Cl⁻ is less distinguishable in X-ray crystallography. Therefore, TR-SFX experiments using I⁻ or Br⁻ are expected to directly visualize the process of ion transport.Here, we report the conformational alterations in NM-R3 during Br⁻ or I⁻ pumping, obtained by both TR-SFX and time-resolved spectroscopy of crystals. The resulting sequence of movements in NM-R3 demonstrates how the chloride pump transports anions with a large ionic radius and prevents the backflow of anions from the cytoplasmic side.