The Effectiveness in Activating M-Type K+ Current Produced by Solifenacin ([(3R)-1-azabicyclo[2.2.2]octan-3-yl] (1S)-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carboxylate): Independent of Its Antimuscarinic Action

Solifenacin (Vesicare®, SOL), known to be a member of isoquinolines, is a muscarinic antagonist that has anticholinergic effect, and it has been beneficial in treating urinary incontinence and neurogenic detrusor overactivity. However, the information regarding the effects of SOL on membrane ionic currents is largely uncertain, despite its clinically wide use in patients with those disorders. In this study, the whole-cell current recordings revealed that upon membrane depolarization in pituitary GH3 cells, the exposure to SOL concentration-dependently increased the amplitude of M-type K+ current (IK(M)) with effective EC50 value of 0.34 μM. The activation time constant of IK(M) was concurrently shortened in the SOL presence, hence yielding the KD value of 0.55 μM based on minimal reaction scheme. As cells were exposed to SOL, the steady-state activation curve of IK(M) was shifted along the voltage axis to the left with no change in the gating charge of the current. Upon an isosceles-triangular ramp pulse, the hysteretic area of IK(M) was increased by adding SOL. As cells were continually exposed to SOL, further application of acetylcholine (1 μM) failed to modify SOL-stimulated IK(M); however, subsequent addition of thyrotropin releasing hormone (TRH, 1 μM) was able to counteract SOL-induced increase in IK(M) amplitude. In cell-attached single-channel current recordings, bath addition of SOL led to an increase in the activity of M-type K+ (KM) channels with no change in the single channel conductance; the mean open time of the channel became lengthened. In whole-cell current-clamp recordings, the SOL application reduced the firing of action potentials (APs) in GH3 cells; however, either subsequent addition of TRH or linopirdine was able to reverse SOL-mediated decrease in AP firing. In hippocampal mHippoE-14 neurons, the IK(M) was also stimulated by adding SOL. Altogether, findings from this study disclosed for the first time the effectiveness of SOL in interacting with KM channels and hence in stimulating IK(M) in electrically excitable cells, and this noticeable action appears to be independent of its antagonistic activity on the canonical binding to muscarinic receptors expressed in GH3 or mHippoE-14 cells.

Hematopoietic Stem Cell Transplantation (HSCT) with Omidubicel Is Associated with Robust Immune Reconstitution and Lower Rates of Severe Infection Compared to Standard Umbilical Cord Blood Transplantation

Introduction Omidubicel is an advanced cell therapy for allogeneic hematopoietic stem cell transplantation (HSCT), derived from appropriately HLA-matched umbilical cord blood (UCB) and comprised of ex-vivo expanded CD133+ cells and a non-cultured lymphocyte-containing fraction. Recent results of a global phase III trial of omidubicel vs standard UCB showed more rapid hematopoietic recovery, reduced rates of infection, and shorter hospitalizations in patients (pts) randomized to omidubicel (Horwitz et al, Blood, 2021). We now report results of correlative immune reconstitution (IR) studies in this trial (NCT02730299). Methods A total of 125 pts aged 13-65 with hematologic malignancies were randomized to allogeneic transplantation with omidubicel or standard UCB following myeloablative conditioning; 108 pts were transplanted per protocol. An optional IR sub-study was conducted and blood was collected at intervals from Day 7 through one year post-transplant. Cryopreserved samples were analyzed in a central laboratory (Covance) using 16-color and 14-color panels and flow cytometric assays to explore T cell, NK cell, B cell, monocyte, and dendritic cell (DC) subsets. Means, medians, ranges, and standard errors were used to summarize cell counts, and one-tailed t-tests were used to compare counts in the two treatment arms. Results A total of 37 pts from 15 sites consented to the IR sub-study, representing 34% of the per protocol population; 17 pts were transplanted with omidubicel and 20 pts with control (15 [75%] of control with double UCB). Median age was 30 (range: 13-62) years for omidubicel pts and 43 (range: 19-55) years for controls in the sub-study. Median CD3+ content of omidubicel prior to cryopreservation was lower (180 x 10^6; range: 71-580 cells) than that of controls post-thaw (516 x 10^6, range: 183-990 cells). Omidubicel pts achieved neutrophil engraftment at a median of 10 (range: 6-28) days post-transplant compared to a median of 18.5 (range: 14-40) days in controls. Omidubicel pts had fewer BMT-CTN Grade 3 viral infections in the first-year post-transplant than controls (6% vs. 25%, respectively). At Day 7 post-transplant, CD4+ T cell counts were significantly higher in omidubicel pts (37x10^3 cells/ml) than in controls (17x10^3 cells/ml, p=0.011). B cells (12x10^3 vs 1x10^3 cells/ml, p=0.013) and NK cells (6x10^3 vs 3 x10^3 cells/ml, p=0.016), as well as monocyte and DC subsets, were also significantly higher in omidubicel pts (Table). Day 14 results similarly demonstrated higher counts of circulating immune cell subsets in omidubicel pts than in controls (Table). Higher B cell counts were observed in omidubicel pts than in controls at 6 months ([863±463] x10^3 vs. [543±221] x10^3 cells/ml, p=0.03) and one year ([1492±370] x10^3 vs [763±150] x10^3, p=0.02) following transplant (Figure). Conclusions Circulating immune cell subsets were consistently higher in omidubicel pts than controls as early as one week after transplant, and higher B cell counts persisted through one year. These findings correlated with the clinical observation of fewer severe bacterial, fungal, and viral infections in pts treated with omidubicel compared to standard UCB. These results demonstrate that rapid hematopoietic recovery in pts transplanted with omidubicel is accompanied by the early and robust appearance of a broad array of lymphocyte, monocyte, DC, and NK cell subsets, despite substantially fewer numbers of these cells infused, suggesting a facilitator effect of omidubicel on their in vivo expansion. Figure 1 Figure 1. Disclosures Szabolcs: Gamida Cell: Consultancy; Prevail Therapeutics: Consultancy; Sotiria/Forge Biologics: Current equity holder in publicly-traded company. Levy: Gamida Cell: Current Employment. Yackoubov: Gamida Cell: Current Employment. Pato: Gamida Cell: Current Employment. Galamidi-Cohen: Gamida Cell, Ltd: Current Employment. Horwitz: Gamida Cell: Research Funding.

Transcriptional and Metabolic Profiling of Nicotinamide-Enhanced Natural Killer (NAM-NK) Cells (GDA-201)

Adoptive transfer of NK cells is a promising immunotherapeutic modality, however limited NK cell persistence and proliferation in vivo have historically been barriers to clinical success. Nicotinamide (NAM), an allosteric inhibitor of NAD-dependent enzymes, has been shown to preserve cell function and prevent differentiation in ex vivo culture of NK (NAM-NK) and other cells. Clinical responses were observed in a Phase 1 trial of NAM-NK (GDA-201) in patients with refractory non-Hodgkin lymphoma (Bachanova, et. al., Blood 134:777, 2019). We now use transcriptional and metabolic profiling to characterize the mechanisms underlying the activity of NAM-NK. CD3 negative lymphocytes obtained from healthy donors were cultured for 14 days with IL-15 in the presence or absence of NAM (7 mM). Next generation sequencing (NGS), liquid chromatography-mass spectrometry (LC-MS)-based metabolite quantification, and glycolytic/mitochondrial respiration measurements were performed. Transcriptome and pathway enrichment analyses were performed with Ingenuity Pathway Analysis software. Extracted cellular and medium metabolites were analyzed on a Thermo Q-Exactive Plus mass spectrometer coupled with a Vanquish UHPLC system. Extracellular acidification (ECAR) and oxygen consumption rates (OCR) were quantified using a Seahorse Extracellular Flux Analyzer. Glycolysis/citric acid cycle (TCA) rates were measured using isotope-labelled glucose incorporation assays. Transcriptome analyses defined 1,204 differentially expressed (DE) genes in NAM-NK vs. control NK. Biological/functional enrichment and pathway analyses of DE-genes predicted upregulation of cell cycle, DNA replication (CDK4/CDKN2D, CyclinD/E, MAD2L), RNA transcription, translation (SMN1/2, ABCF1, EIF4B, RPL13, RPS6), protein synthesis (EIF2, PABPC1, SOS, 60S complex) mitochondrial energy metabolism (NDUFB8, ATP5G2/E, COX7B/C) migration, homing (CD62L, CD44, DNAM1), and cytokine/chemokine response (IL18R, CXCR3, CCR5, XCL1, SOCS3, LFA1) pathways, with concomitant downregulation of cell exhaustion, senescence (BATF1, FOXP1, STAT1, CD86, LGALS9, LAG3), apoptosis, necrosis (CASP1, MDM2, IKK3), stress response (CALR, HSP90, HSPH1), and lymphoid cellular maturation (IL-2Ra, CD40L, GATA3) pathways in NAM-NK. Metabolomic analyses showed a significant increase of intracellular NAD, NADH, NADP, NADPH, high-energy triphosphates (ATP, UTP, GTP) and overall energy charge ([ATP+0.5*ADP]/[ATP+ADP+AMP]) in NAM-NK. Cellular metabolic fitness analyses revealed increased basal and ATP-linked respiration, mitochondrial maximal respiratory capacity, and glycolytic capacity in NAM-NK compared to control NK. In addition, NAM increased the rate of glucose incorporation into TCA cycle intermediates (acetyl-CoA, succinyl-CoA), consistent with a more rapid glycolysis rate, increased TCA cycling, and improved glucose consumption efficiency. Taken together, results of transcriptome, metabolomic, mitochondrial respiration, and glycolytic rate analyses suggest that NAM pleiotropically modulates key cellular metabolic functions in ex vivo-expanded NK cells, resulting in increased response to cytokine stimulation and enhanced potency. NAM inhibits differentiation, cellular stress, and exhaustion pathways that are typically activated in culture. Moreover, NAM increases cellular metabolic fitness, energy charge, and efficiency of glucose consumption, potentially imparting a protective effect against oxidative stress in the tumor microenvironment. These data offer insight into the mechanism of improved persistence, proliferation, and cytotoxicity observed in in vivo and clinical studies of GDA-201. Disclosures Yackoubov: Gamida Cell: Current Employment. Pato: Gamida Cell: Current Employment. Rifman: Gamida Cell: Current Employment. Cohen: Gamida Cell: Current Employment. Hailu: Gamida Cell: Current Employment. Persi: Gamida Cell: Current Employment. Berhani-Zipori: Gamida Cell: Current Employment. Edri: Gamida Cell: Current Employment. Peled: Biokine Therapeutics Ltd: Current Employment; Gamida Cell: Research Funding. Cichocki: Gamida Cell: Research Funding; Fate Therapeutics, Inc: Patents & Royalties, Research Funding. Rabinowitz: Gamida Cell: Research Funding. Lodie: Gamida Cell: Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Adams: Gamida Cell: Current Employment. Simantov: Gamida Cell: Current Employment. Geffen: Gamida Cell: Current Employment.

Remodeling of ryanodine receptor isoform 1 channel regulates the sweet and umami perception of Rattus norvegicus

Sweet and umami are respectively elicited by sweet/umami receptor on the tongue and palate epithelium. However, the molecular machinery allowing to taste reaction remains incompletely understood. Through a phosphoproteomic approach, we found the key proteins that trigger taste mechanisms based on the phosphorylation cascades. Thereinto, ryanodine receptor isoform 1 (RYR1) was further verified by sensor and behaviors assay. A model proposing RYR1-mediated sweet/umami signaling: RYR1 channel which mediates Ca2+ release from the endoplasmic reticulum is closed by its dephosphorylation in the bud tissue after umami/sweet treatment. And the alteration of Ca2+ content in the cytosol induces a transient membrane depolarization and generates cell current for taste signaling transduction. We demonstrate that RYR1 is a new channel in regulation of sweet/umami signaling transduction and also propose a metabolic clock notion based on sweet/umami sensing. Our study provides a rich fundamental for a system-level understanding of taste perception mechanism.

Pre-Emphasis Pulse Design for Random-Access Memory

This paper describes how one can reduce the memory access time with pre-emphasis (PE) pulses even in non-volatile random-access memory. Optimum PE pulse widths and resultant minimum word-line (WL) delay times are investigated as a function of column address. The impact of the process variation in the time constant of WL, the cell current, and the resistance of deciding path on optimum PE pulses are discussed. Optimum PE pulse widths and resultant minimum WL delay times are modeled with fitting curves as a function of column address of the accessed memory cell, which provides designers with the ability to set the optimum timing for WL and BL (bit-line) operations, reducing average memory access time.

Transient incubation of cultured hippocampal neurons in the absence of magnesium induces rhythmic and synchronized epileptiform-like activity

Cell culture models are important tools to study epileptogenesis mechanisms. The aim of this work was to characterize the spontaneous and synchronized rhythmic activity developed by cultured hippocampal neurons after transient incubation in zero Mg2+ to model Status Epilepticus. Cultured hippocampal neurons were transiently incubated with a Mg2+-free solution and the activity of neuronal networks was evaluated using single cell calcium imaging and whole-cell current clamp recordings. Here we report the development of synchronized and spontaneous [Ca2+]i transients in cultured hippocampal neurons immediately after transient incubation in a Mg2+-free solution. Spontaneous and synchronous [Ca2+]i oscillations were observed when the cells were then incubated in the presence of Mg2+. Functional studies also showed that transient incubation in Mg2+-free medium induces neuronal rhythmic burst activity that was prevented by antagonists of glutamate receptors. In conclusion, we report the development of epileptiform-like activity, characterized by spontaneous and synchronized discharges, in cultured hippocampal neurons transiently incubated in the absence of Mg2+. This model will allow studying synaptic alterations contributing to the hyperexcitability that underlies the development of seizures and will be useful in pharmacological studies for testing new drugs for the treatment of epilepsy.

Modulation of recombinant human alpha 1 glycine receptor by flavonoids and gingerols

The inhibitory glycine receptor (GlyR) is a principal mediator of fast synaptic inhibition in mammalian spinal cord, brainstem, and higher brain centres. Flavonoids are secondary plant metabolites that exhibit many beneficial physiological effects, including modulatory action on neuronal receptors. Using whole-cell current recordings from recombinant human α1 GlyRs, expressed in HEK293 cells, we compared the flavonols kaempferol and quercetin, the flavanone naringenin, the flavones apigenin and nobiletin, the isoflavone genistein, and two gingerols, 6-gingerol and 8-gingerol for their modulation of receptor currents. All compounds were inhibitors of the GlyR with IC50 values ranging between 9.3 ± 2.6 µM (kaempferol) and 46.7 ± 6.5 µM (genistein), following a mixed mode of inhibition. Co-application of two inhibitors revealed distinct binding sites for flavonoids and gingerols. Pore-lining mutants T258A and T258S were strongly inhibited by quercetin and naringenin, but not by 6-gingerol, confirming the existence of distinct binding sites for flavonoids and gingerols. Apigenin, kaempferol, nobiletin, naringenin and 6-gingerol showed biphasic action, potentiating glycine-induced currents at low concentration of both, modulator and glycine, and inhibiting at higher concentrations. Identification of distinct modulatory sites for flavonoids and related compounds may present pharmacological target sites and aid the discovery of novel glycinergic drugs.