Pharmacological Research

There is a pressing need for effective alternatives to opioid analgesics, the development of which requires the identification of novel anti-nociceptive drug targets. Here, we have further investigated the anti-nociceptive properties of a GPR35 agonist, cromolyn, in an in vitro model of inflammatory sensitisation. We used ratiometric Ca2+ imaging of cultured sensory neurons to examine the effect of cromolyn on prostaglandin E2 (PGE2)-mediated sensitisation of the pro-nociceptive ion channel, transient receptor potential cation channel, subfamily V, member 1 (TRPV1). The sensitisation of TRPV1 by PGE2 was inhibited by cromolyn in a GPR35-dependent manner. These observations provide further evidence in support of an anti-nociceptive role for GPR35, highlighting the potential use of GPR35 agonists as analgesics.

BACKGROUNDMyocardial ischemia/reperfusion injury (I/RI) represents a serious clinical complication in patients after acute myocardial infarction. Ubiquitin-activating enzyme 1 (UBA1) catalyzes the initial step of ubiquitination and plays a fundamental role in regulating protein homeostasis and related diseases. This study aims to elucidate the functional contribution of UBA1 to the pathogenesis of myocardial I/RI and to uncover its underlying mechanisms. METHODSSingle-cell RNA sequencing was employed to characterize UBA1 expression in human ischemic heart tissues. Myocardial I/R injury was examined in myocardial-specific UBA1 knockout (UBA1cko) mice, UBA1-overexpressing mice (rAAV9-UBA1), and corresponding controls. Neonatal rat cardiomyocytes underwent hypoxia/reoxygenation in vitro. Cardiac function and infarction were evaluated by echocardiography and pathological staining. Protein-protein interactions were analyzed via immunoprecipitation combined with mass spectrometry. The endoplasmic reticulum-mitochondrial contact sites (ERMCSs) and mitochondrial ultrastructure were evaluated through transmission electron microscopy and confocal imaging. RESULTSUBA1 expression was significantly downregulated in human and murine ischemic myocardium, especially in cardiomyocytes. UBA1cko mice exhibited aggravated I/RI with greater infarct size, impaired function, apoptosis, elevated intracellular Ca2+ levels, mitochondrial dysfunction, and ER stress, whereas UBA1 overexpression conferred cardioprotective effects. Mechanistically, UBA1 directly bound to and ubiquitinated Pdzd8, a key ERMCS-tethering protein, thereby promoting its degradation, which inhibited ERMCS formation and improved mitochondrial dysfunction and ER stress. Moreover, knockdown of Pdzd8 via rAAV9-siRNA effectively mitigated UBA1 knockout-induced myocardial damage. Additionally, administration of auranofin (AF), a U.S. Food and Drug Administration-approved drug for treating rheumatoid arthritis, markedly alleviated myocardial I/RI via activating UBA1 in vivo and in vitro. CONCLUSIONSUBA1 confers protection against myocardial I/RI by limiting ERMCS formation through Pdzd8 ubiquitination. Activating UBA1 or targeting Pdzd8 as a potential therapeutic strategy for the treatment of ischemic heart disease. GRAPHIC ABSTRACTA graphic abstract is available for this article. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIUBA1 expression is downregulated in human and murine ischemic myocardium, especially in cardiomyocytes. C_LIO_LICardiac deletion of UBA1 significantly exacerbates myocardial ischemia/reperfusion injury (I/RI), whereas cardiac UBA1 overexpression confers a marked protective effect. C_LIO_LIUBA1 interacts with Pdzd8 (PDZ domain containing 8) and facilitates its ubiquitination and subsequent degradation, which then reduces endoplasmic reticulum-mitochondria contact sites (ERMCSs) and ameliorates mitochondrial dysfunction and ER stress, protecting myocardial I/RI. C_LIO_LIPharmacological activation of UBA1 with the FDA-approved drug auranofin attenuates myocardial I/R injury and improves heart dysfunction. C_LI What Are the Clinical Implications?O_LIUBA1 represents a new therapeutic target for myocardial I/RI. C_LIO_LIActivating UBA1 or targeting Pdzd8 may offer a promising therapeutic strategy for mitigating myocardial I/RI and heart failure, underscoring its potential for clinical translation. C_LI

Neuroactive steroids modulate GABAA and NMDA receptors allosterically, typically requiring specific structural features for their activity. In this study, we characterize YX84, a novel neuroactive steroid bearing a 3{beta} sulfate and p-trifluoroacetylbenzyl alcohol attached in an ether linkage to a hydroxyl group at steroid carbon 17. This compound and similar analogues exhibit an atypical pharmacological profile, with three distinct actions at GABAA receptors. First, YX84 is a full agonist, with EC50 near 1 {micro}M and comparable efficacy to GABA at GABAA receptors in native hippocampal neurons. It presents as a full agonist relative to GABA at 4/{delta} subunit-containing receptors. Second, YX84 acts as a slow-onset, potent positive allosteric modulator (PAM) of GABAA receptors at concentrations below those that gate a response. Finally, YX84 exhibits rapid desensitizing and/or blocking kinetics; voltage dependence is consistent with a contribution of channel block. Structure- activity relationship analyses reveal that both functional groups are essential for gating activity, while classical requirements such as carbon 3 hydroxyl stereoselectivity and carbon 5 reduction are dispensable. YX84 also modestly inhibits NMDA receptor currents, suggesting weak negative allosteric modulation. Behavioral assays show that intraperitoneal administration of YX84 (30 mg/kg) does not impair sensorimotor function, unlike allopregnanolone. These findings identify YX84 as a structurally distinct neuroactive steroid with dual receptor activity and favorable behavioral tolerability, offering a promising scaffold for therapeutic development targeting excitatory/inhibitory imbalance in neuropsychiatric disorders if pharmacokinetic considerations can be overcome.

Fingolimod (FNG) is a sphingosine-1-phosphate receptor agonist currently prescribed for the treatment of remitting-relapsing multiple sclerosis. However, an increasing body of evidence indicates that FNG has a variety of other effects on the central nervous system, making it a good candidate to target other brain disorders that display loss of neuronal cells and synaptic plasticity. FNG treatments induce production of brain-derived neurotrophic factor (BDNF), which promotes neuronal plasticity, arborization and survival via signaling through its cognate receptor TRKB. In this study we characterize the relationship between FNG and TRKB in vitro and in vivo following acute treatments. We found that FNG induces TRKB activation in primary neuronal cultures in a BDNF-dependent way, indicating a rapid effect of FNG. This effect is different from the one elicited by antidepressants and is likely mediated by modulation of plasma membrane properties, as the enhancement of fluoxetine binding and dimerization of the cholesterol-insensitive TRKB mutant Y433F mimic the effects of cholesterol. Moreover, acute FNG treatment normalizes the generalization of conditioned fear response seen in heterozygous BDNF null female mice without affecting the wild-type littermates. Taken together, our data indicate that FNG allosterically promotes TRKB signaling and thereby induces the increase in BDNF production, which mediates the therapeutic effects of the drug on neuronal plasticity.

The immune checkpoint receptor PD-1, traditionally known for suppressing T-cell activation, is also expressed in peripheral sensory neurons where it limits excitability, but the physiological contexts in which neuronal PD-1 is engaged remain poorly defined. We previously identified a purified Staphylococcus epidermidis lipoteichoic acid analog (SELTA) that rapidly alleviates pelvic pain in mice. Here, we investigated whether SELTA functions as an exogenous activator of neuronal PD-1 and whether PD-1 is required for its analgesic effects. SELTA increased Pdcd1 transcripts and PD-1 protein in dorsal root ganglion (DRG) neurons and prostate-innervating sensory fibers, and colocalized with TLR2 at neuronal membranes where it enhanced PD-1 Y248 phosphorylation. In primary DRG cultures, SELTA suppressed ATP-evoked Ca{superscript 2} responses, indicating reduced neuronal excitability. This effect was abolished by PD-1 neutralization but restored when neutralization was blocked by a PD-1 control peptide, demonstrating both PD-1 dependence and specificity. In male mice with chronic pelvic allodynia induced by experimental autoimmune prostatitis, intraurethral SELTA produced robust, concentration-dependent, and repeatable reductions in tactile hypersensitivity. Sensory neuron-specific PD-1 conditional knockout mice showed normal baseline behavior but failed to respond to SELTA, confirming that PD-1 expression in nociceptors is essential for SELTA-mediated analgesia. These findings identify SELTA as a commensal-derived activator of neuronal PD-1 signaling. Through TLR2 engagement, PD-1 phosphorylation, and suppression of excitatory calcium signaling, SELTA attenuates nociceptor activation and reduces pelvic pain. This mechanism highlights neuronal PD-1 as a modifiable checkpoint pathway and a promising non-opioid target for chronic pain therapy.

Major Depressive Disorder (MDD) is increasingly conceptualized as a neuroimmune-metabolic-oxidative stress (NIMETOX) condition, partly driven by environmental stressors. While new antidepressant strategies have emerged to target NIMETOX pathways, the mechanism by which chronic stress reshapes metabolic regulation-particularly in the absence of clinically overt metabolic disease-remain poorly understood. Using a 6-week chronic unpredictable mild stress (CUMS) paradigm in metabolically healthy male and female mice, we evaluated depressive-like behaviors alongside circulating glucose and metabolic hormones, including insulin, resistin, Glucose-dependent Insulinotropic Polypeptide (GIP), Glucagon-like Peptide-1 (GLP-1), glucagon, ghrelin, leptin, and Plasminogen Activator Inhibitor-1 (PAI-1). We further assessed whether simvastatin, curcumin, S-adenosyl methionine (SAMe), or pyrrolidine dithiocarbamate (PDTC), an NF-{kappa}B inhibitor, normalize CUMS-induced metabolic and behavioral alterations, using fluoxetine as a reference antidepressant. CUMS induced a hypoinsulinemic state accompanied by enhanced basal insulin sensitivity and reduced insulin resistance, while central appetite regulation and adipose inflammatory profiles remained preserved. Indices of lower insulin resistance were strongly associated with sucrose preference and immobility time. Glucagon and PAI-1 showed positive associations with novel object recognition performance, whereas GIP and leptin were inversely related to open-field activity. Glucose levels correlated positively with rearing behavior. Despite significant improvements in depressive-like behaviors, none of the pharmacological interventions normalized the stress-induced metabolic hormone profile. Marked sex differences were observed, with females displaying a catabolic, immune-ready phenotype and males showing a relative anabolic bias. These findings indicate that CUMS induces a non-pathological, adaptive metabolic hormone reprogramming rather than metabolic dysfunction, supporting the interpretation of stress-related metabolic changes as resilient adaptations within the NIMETOX framework.

Repeated exposure to hypoxia (oxygen levels below sea-level atmospheric conditions, [~]21%) alternated with regular voluntary exercise, known colloquially as Living High, Training Low, or simply High-Low, is used by elite athletes to boost exercise benefits and athletic performance. While paradigms of High-Low training have been utilized by Olympic athletes for decades, the therapeutic potential of a High-Low regimen in the context of neurotrauma has yet to be investigated. This long-term experiment evaluated the independent and combined effects of repeated hypoxic exposure and voluntary exercise on functional outcomes within the context of preclinical spinal cord injury (SCI). We hypothesized that combinatorial High-Low training enhances functional recovery, beyond either exercise or repeated exposures to hypoxia alone, to improve outcomes after SCI. Adult female rats (n=62) underwent a high-cervical hemisection (LC2H) to model spinal cord injury. At 6 weeks post-SCI, treatment (access to exercise wheel, repeated exposure to normobaric hypoxia at rest, or alternation of both) began in the surviving subjects (n=49). Despite initiation of treatment beyond the acute post-injury phase, High-Low therapy significantly improved respiratory function and prevented the development of SCI-associated anxiety-like behaviors. Notably, repeated in vivo exposure to normobaric hypoxia induced a shift in peripheral T cell profiles, characterized by increased CD4+ and reduced CD8+ expression. These findings indicate that combining repeated exposure to hypoxia with voluntary exercise as a therapy could promote recovery in the existing spinal cord-injured population. Collectively, this work provides a foundational first step for further investigation of High-Low training as a rehabilitation therapy for individuals living with SCI.

Anorexia nervosa (AN) is a debilitating, often lethal, restrictive-type eating disorder without an effective cure. The underlying neural basis of AN has remained elusive without an animal model that has represented all typical AN symptoms. Here we show that aberrant activation of mediobasal hypothalamic (MBH) glutamatergic neurons led to lethal self-starvation, hyperactivity, anhedonia, social phobia, and increased anxiety, all of which represent typical symptoms of AN. These symptoms were selectively exhibited by targeted activation of MBH neurons expressing steroidogenic factor (SF1) and estrogen receptor alpha (ERa). Moreover, the elicited AN symptoms by activation of MBH glutamatergic or SF1/ERa neurons were rescued by removing release of glutamate or brain-derived neurotrophic factor (BDNF) from these neurons. Importantly, BDNF overexpression in SF1/ERa neurons promoted typical AN symptoms, which were suppressed by removing glutamate release. Thus, our findings identify aberrantly enhanced BDNF and consequent augmented glutamate release from SF1/ERa neurons as a neural basis underlying AN.

SGLT2 inhibitor (SGLT2i)-induced diabetic hyperketonemia is a life-threatening acute complication of diabetes. While Celastrol has been reported to exert beneficial effects on obesity; its potential role in ketogenesis remains unclear. In this study, Celastrol administration significantly attenuates the fasting-induced elevation of blood {beta}-hydroxybutyrate. Moreover, a 7-day course of Celastrol (1 mg/kg/day) leads to reductions in body weight and fat mass. Mechanistically, Celastrol specifically downregulates HMGCS2 expression and suppressess hepatic ketogenesis through inhibiting PPAR expression in the short term ([≤] 2 days). However, after prolonged treatment for 7 days, Celastrol modulates both PPARand serum free fatty acids (FFAs) levels. Furthermore, anti-ketogenic effect of Celastrol is abolished in Ppar{square} /{square} mice. Importantly, Celastrol effectively ameliorates SGLT2i-induced hyperketonemia. In summary, Celastrol curbs hepatic ketone overproduction in a PPAR-dependent manner, indicating its protective potential against SGLT2i-induced hyperketonemia.

Control of synaptic transmission efficacy by neuronal activity and neuromodulators is pivotal for brain function. Synaptic suppression by cannabinoids activating CB1 receptors has been extensively studied at the molecular and cellular levels to understand the neuronal basis for effects of cannabis intake. Here, we focused on GPR55, non-canonical type of cannabinoid receptor, which shows sensitivity to cannabidiol included in cannabis, aiming to highlight its actions on presynaptic function. Taking advantage of direct patch-clamp recordings from axon terminals of cerebellar Purkinje cells together with fluorescent imaging of vesicular exocytosis using synapto-pHluorin, we show that GPR55 suppresses synaptic transmission as CB1 receptor does, but through a distinct presynaptic modulation of release machinery. Activation of GPR55 reduced transmitter release by changing neither presynaptic action potential waveform nor Ca2+ influx, but by making a large population of Ca2+-responsive synaptic vesicles insensitive to Ca2+ influx through voltage-gated Ca2+ channels, leading to substantial reduction of the readily releasable pool of vesicles. Thus, the present study identifies a unique mechanism to suppress presynaptic transmitter release by an atypical cannabinoid receptor GPR55, which would enable subtype-specific modulation of neuronal computation by cannabinoid receptors.

Class B scavenger receptors BI (SR-BI) and BII (SR-BII) internalize lipoproteins but also bind and internalize bacteria. Their roles in sepsis are unknown. We overexpressed human SR-BI and BII in the liver and kidney as well as bone marrow-derived macrophages, and then performed cecal ligation and puncture (CLP) surgery. SR-BI and BII transgenic mice had significantly worse survival compared to WT mice. 24 h after CLP, liver injury markers and histological damage were prominent in both SR-BI and BII transgenic mice, whereas kidney damage was similar. Systemic inflammatory cytokines were markedly increased in SR-BI and BII transgenic mice; parallel increases were seen in liver mRNA expression, not in the kidney. The highest degree of neutrophil infiltration was observed in the liver of SR-BI. Human SR-BI and BII dramatically decreased bacterial accumulation in the liver. Green fluorescence protein-labeled E. coli were efficiently phagocytosed in hepatic macrophages of SR-BI and BII transgenic mice; phagocytosis was more prominent in SR-BII transgenic mice. Finally, human SR-BI overexpression reduced systemic HDL-C level, eliminated adrenal cortex lipid droplets, and dampened the systemic increase of corticosterone after CLP. Supplementation with glucocorticoid and mineralocorticoid improved survival in SR-BI, but not SR-BII, transgenic mice after CLP. In summary, our findings suggest human SR-BI and BII overexpression contributes to higher mortality after CLP by excessive inflammatory response due to adrenal insufficiency (SR-BI) or hyperactive phagocytosis (SR-BII) in the liver.

BackgroundParkinsons disease (PD) is a progressive chronic neurodegenerative disease. The PARK2 gene encoding the Parkin protein accounts for approximately half of early-onset autosomal recessive PD cases in humans. ObjectiveThe aim of this work was to study the effect of the PARK2 gene knockout in mice on the dynamics of behavioral and biochemical parameters of PD. MethodsThe study was performed on C57BL/6-line mice aged from 4 months to 1.5 years: wild type (park2 +/+), heterozygotes (park2 +/-) and homozygotes (park2 -/-) knocked out by the PARK2 using CRISPR-Cas9. The open field test, the Porsolt forced swimming test, the grid-walk test, the beam-walking test, the elevated plus maze test, the accelerating rotarod test were used to assess the behavioral phenotype. Measurement of the concentration of bioamines and their metabolites by HPLC and evaluation of the amount of tyrosine hydroxylase, BDNF and GDNF by Western Blot were used to study the biochemical signs of PD. ResultsPark2 -/- mice begin to show signs of decreased motor activity no earlier than at 4 months of life. At 12 months of life, it was shown only a decrease in the level of the mature isoform of GDNF and an increase in the number of immature isoforms in the frontal cortex and striatum were revealed. ConclusionThe data obtained indicates a different age dynamic of the condition of mice associated with the PARK2 knockout. However, no pronounced specific manifestations of PD in human were found in park2 -/- mice.

Sepsis is defined as a dysregulated response to infection, leading to life-threatening organ dysfunction that particularly affects parenchymal organs. Clinical studies remain inconclusive regarding the impact of biological sex on sepsis, and preclinical studies are predominantly performed in male animals. We examined early (8 h) septic responses in male and female mice using a fecal-induced peritonitis (FIP) model. Blood biochemical parameters, body temperature, and murine sepsis scores provided evidence of a septic response in animals randomized to FIP compared to controls, but showed no physiological differences between male and female mice. Transcriptomic analysis of the liver, kidney, and lung showed consistent inflammatory activation in response to sepsis as compared to controls. Notably, in the kidney and lung, female mice exhibited stronger immune activation and a heightened inflammatory response compared to males. Thus, biological sex differences in the septic response can be detected in early acute sepsis without apparent physiological differences.

Excessive osteoclast formation is a key contributor to pathological bone loss in disorders such as osteoporosis and rheumatoid arthritis. Asarinin, a natural lignan, has not previously been examined in the context of osteoclast differentiation. Here, we investigated the anti-osteoclastogenic effects of asarinin using RANKL-stimulated RAW264.7 cells. Asarinin significantly suppressed TRAP-positive multinucleated osteoclast formation under the tested conditions. Mechanistically, asarinin selectively inhibited RANKL-induced phosphorylation of p38 and ERK MAPKs, leading to reduced c-Fos expression and inhibition of NFATc1 nuclear translocation. In addition, asarinin disrupted actin ring formation in mature osteoclasts. Collectively, these findings identify asarinin as a pathway-selective inhibitor of osteoclast differentiation, targeting the p38/ERK-c-Fos-NFATc1 axis while sparing parallel signaling pathways.

BackgroundAnthracyclines such as doxorubicin are effective chemotherapeutics but are limited by cardiotoxicity driven in part by mitochondrial dysfunction. Dysregulated mitochondrial dynamics, particularly excessive dynamin-related protein-1 (Drp1)-mediated fission, contribute to doxorubicin-induced cardiac injury and support selective survival of cancer cells. ObjectivesTo determine whether DRP1i2, a novel small molecule Drp1 inhibitor targeting a conserved domain shared between human and mouse, can function as a cardio-oncology therapeutic by reducing doxorubicin-induced cardiotoxicity while maintaining or enhancing anti-cancer efficacy. MethodsCardioprotective effects of DRP1i2 were evaluated in a murine model of chronic doxorubicin cardiotoxicity and in human induced pluripotent stem cell-derived cardiac microtissues exposed to acute doxorubicin injury. Anticancer activity was assessed across multiple cancer cell lines using 2D monolayers and 3D microtissues. ResultsIn vivo, DRP1i2 preserved left ventricular ejection fraction, reduced interstitial fibrosis and cardiomyocyte atrophy, and attenuated doxorubicin-induced myocardial proteomic remodelling. In human cardiac microtissues, DRP1i2 improved viability and restored contractile function despite persistent mitochondrial oxidative stress. DRP1i2 showed modest anticancer activity in MG63 osteosarcoma cells in both 2D and 3D systems and did not diminish doxorubicin efficacy in other cancer models (MDA-MB-231 breast, OVCAR3 ovarian, and A549 lung adenocarcinoma). Combined treatment further enhanced cytotoxicity selectively in MG63 cells. ConclusionsDRP1i2 exerts complementary cardioprotective and anticancer actions through modulation of shared mitochondrial pathways, identifying Drp1 as a druggable target in cardio-oncology. These findings support DRP1i2 as a first-in-class Drp1 inhibitor and highlight mitochondrial dynamics as a promising therapeutic axis to preserve anthracycline efficacy while reducing cardiotoxicity. Clinical PerspectivesExcessive Drp1-mediated mitochondrial fission links anthracycline cardiotoxicity with cancer cell survival. Inhibition with DRP1i2 preserved cardiac structure and function in a chronic doxorubicin cardiotoxicity model without compromising anti-cancer activity, representing mechanism-based cardioprotection, where the heart is protected by directly targeting the molecular processes driving injury. Translation will require pharmacologic profiling and testing in tumour-bearing and comorbid models, followed by early-phase trials to confirm safety and efficacy.

Parkinsons disease (PD) is the second most common neurodegenerative disease, resulting from accumulation of -synuclein (-syn) in midbrain dopaminergic neurons and progressive neuronal loss. The most relevant species of -syn, oligomers, may exert neurotoxicity in a variety of mechanisms. Accumulation of misfolded -syn in the endoplasmic reticulum (ER) lumen induces ER stress conditions that leads to activation of the Unfolded Protein Response (UPR) and its main sensor PKR-like ER kinase (PERK). PERK is critical for cell fate determination - under prolonged ER stress, it may direct cell towards pro-apoptotic pathways. Targeting of -syn aggregation or UPR by genetic and pharmacological approaches proved effective in preclinical models of PD by previous research. Thus, in the present study, we aimed to determine the potential effect of combination of small-molecule inhibitors of -syn aggregation and ER stress-mediated PERK signaling (namely anle138b and AMG44) in a novel, 3D in vitro model of PD. We demonstrate that combination of both anti-aggregation and ER stress-targeting approaches amplifies neuroprotection against PD in organoid model in terms of increased neuronal metabolic activity, decreased -syn phosphorylation and aggregation, reduced dopaminergic cell death, and restoration of proteostasis.

Background: Potassium is involved in multiple physiological processes in the body, and hyperkalemia is a common, potentially life-threatening condition. Objective: The aim of our study was to examine the association between plasma potassium levels, and 30-day mortality in patients presenting to an emergency department with normo- or hyperkalemia. Design: Retrospective Cohort study. Setting: Emergency Departments in the Capital region of Denmark Participants: Persons attending Emergency Departments in the Capital Region of Denmark from 2017--2021 with a plasma potassium level of at least 3.5 mM measured within 4 hours after arrival. Measurements: The study was based on data from Danish National Registries and electronic patient records. We performed Kaplan-Meier survival analyses and unadjusted and adjusted cox regression analyses utilizing plasma [K+] 3.5--4.4 mM as the reference group for 30-day mortality hazard ratios (HRs). Results: A total of 248,453 patients were included with a median age of 60 years (Q1;Q3 42;75), and 6,959 (2.8%) died within 30 days. Mortality was 2.2% for potassium level 3.5--4.4 mM, 6.9% for 4.5--4.9 mM, 17.1% for 5.0--5.9 mM, and 26.9% for [≥] 6.0 mM. Unadjusted 30-day HRs were 3.2 (95%CI: 3.0--3.4) for [K+] 4.5--4.9 mM, 8.6 (95%CI: 7.9--9.3) for [K+] 5.0--5.9 mM, and 14.7 (95%CI: 12.5--17.0) for [K+] [≥]6.0 mM. Adjusted HRs were 1.4 (1.3--1.5), 2.10 (1.9--2.3), and 2.4 (2.0--2.8), respectively. Limitations: Risk of residual confounding. Missing data. No access to data regarding in-hospital treatment. Conclusion: Plasma potassium levels above 4.4 mM were associated with increased 30-day mortality among patients presenting to emergency departments. Primary funding source: Department of Emergency Medicine, Copenhagen University hospital, Bispebjerg and Frederiksberg Hospital.

Amyotrophic Lateral Sclerosis (Lou Gehrigs disease) is a progressive neurodegenerative disease affecting hundreds of thousands of people worldwide. It is characterized by the degeneration of the neurons in the brain and spinal cord of the patients, leading to a loss of control of muscles. Over time, without nerves to stimulate them muscles tend to atrophy. ALS may occur sporadically or run in families; many mutations have been identified for the latter. Treatment of ALS is mostly limited to three approved therapeutic agents: riluzole, edaravone, and tauroursidiol/ sodium phenylbutyrate. Among these, riluzole remains the most effective despite its early discovery. There are no conclusive meta-analysis comparing riluzole monotherapy to all possible co-therapies present. In this work we have attempted to address such a concern and observed that no adjunct therapy significantly improved the performance of riluzole. However, mitochondrial/ oxidative stress modulator and neuroimmune/ neuroexcitability modulator co-therapy exhibited positive trends. Surprisingly, trials were mainly confined to the USA and European countries, indicating unequal demographic representation in ASL research. We have concluded that large double blinded inter-continental RCTs to be carried out for better understanding of the scenario.

Sensory transmission in the monosynaptic stretch reflex (MSR) from proprioceptive Ia afferents to motoneurons is markedly inhibited by serotonin, providing a mechanism by which the brain gates spinal cord function. However, the main serotonin receptor subtype that modulates the MSR is the 5-HT1D receptor that is expressed almost exclusively on nociceptive C-fibres, suggesting an unexpected gating of proprioceptive transmission by regulation of nociceptive pathways. We demonstrate here that activation of C-fibres (electrically or pharmacologically by TRPM8 agonists) increases action potential propagation in proprioceptive Ia afferents by producing a very long-lasting tonic depolarization in Ia afferents (tonic primary afferent depolarization, tonic PAD). This tonic PAD is produced indirectly by C-fibres augmenting axoaxonic GABAergic input onto Ia afferents (bicuculline sensitive), which activates nodal GABAA receptors that aid sodium spike initiation. In contrast, activation of 5-HT1D receptors with the anti-migraine drug zolmitriptan inhibits C-fibre activity, which in turn reduces the GABAA receptor-mediated tonic PAD on Ia afferents, and reduces spike propagation to motoneurons. Overall, these findings demonstrate a mechanism by which brainstem derived serotonin not only inhibits ongoing nociceptive C-fibres activity, but also indirectly inhibits GABAergic neurons and sensory transmission in large proprioceptive sensory afferents, leading to reduced motoneuron activity. Furthermore, these findings suggest that spontaneous nociceptive C-fibres activity may provide a general increase in proprioception, opposite to the classical gating of nociceptive pathways by low threshold afferents, and impacting the clinical use of zolmitriptan.

Chronic pain affects a significant portion of the global population and imposes substantial clinical and socioeconomic burdens. Current treatments mainly rely on opioid analgesics, which carry serious risks of dependence and misuse, underscoring the urgent need for alternative therapeutic strategies. Galanin receptors (GALR1-3) are known to be involved in modulating pain, yet their specific roles remain poorly understood due to the lack of receptor subtype-selective ligands. Recently, spexin has been identified as an endogenous peptide that selectively activates GALR2 and GALR3, offering a new scaffold for developing pharmacological tools targeting these receptor subtypes. In this study, we report the design and characterization of a modified spexin analog, LIT-01-144, engineered through N-terminal functionalization with a fluorocarbon chain to improve metabolic stability while preserving receptor selectivity. In vitro assays showed that LIT-01-144 has high potency at GALR2 and GALR3, with minimal activity at GALR1. Pharmacokinetic studies revealed a significantly longer plasma half-life compared to native spexin and no central nervous system penetration. In mice, intracerebroventricular administration of LIT-01-144 produced strong antinociceptive effects at doses ten times lower than spexin. While systemic administration showed no notable antinociception in naive animals, LIT-01-144 significantly reduced pain responses in a mouse model of persistent inflammatory pain induced by complete Freunds adjuvant (CFA). This antinociceptive activity was specifically mediated through GALR2 and was independent of opioid receptor pathways. In situ hybridization further showed an increase in Galr2-positive neurons in dorsal root ganglia of inflamed mice. Overall, these findings highlight GALR2 as a promising peripheral target for developing non-opioid analgesics and demonstrate the potential of LIT-01-144 as a valuable tool for understanding GALR2-mediated mechanisms of pain modulation.