Inhibition Of Response Research Articles

Two-photon imaging of excitatory and inhibitory neural response to infrared neural stimulation.

Pulsed infrared neural stimulation (INS, 1875nm) is an emerging neurostimulation technology that delivers focal pulsed heat to activate functionally specific mesoscale networks and holds promise for clinical application. However, little is known about its effect on excitatory and inhibitory cell types in cerebral cortex. Estimates of summed population neuronal response time courses provide a potential basis for neural and hemodynamic signals described in other studies. Using two-photon calcium imaging in mouse somatosensory cortex, we have examined the effect of INS pulse train application on hSyn neurons and mDlx neurons tagged with GCaMP6s. We find that, in anesthetized mice, each INS pulse train reliably induces robust response in hSyn neurons exhibiting positive going responses. Surprisingly, mDlx neurons exhibit negative going responses. Quantification using the index of correlation illustrates responses are reproducible, intensity-dependent, and focal. Also, a contralateral activation is observed when INS applied. In sum, the population of neurons stimulated by INS includes both hSyn and mDlx neurons; within a range of stimulation intensities, this leads to overall excitation in the stimulated population, leading to the previously observed activations at distant post-synaptic sites.

Predictors of neuromodulation during single-shot thermal atrial fibrillation ablation: a computed tomography imaging analysis

Abstract Background Ganglionated plexi (GPs) are part of the cardiac autonomic nervous system (CANS), which play an important role in the induction and maintenance of atrial fibrillation (AF). Because of the anatomic proximity of the pulmonary veins with GPs, pulmonary vein isolation (PVI) using thermal (radiofrequency (RF) or cryo) ablation, has been shown to concomitantly affect the GPs, as suggested by increased heart rate (HR) after ablation. Right superior pulmonary vein (RSPV) isolation has been reported to have the most evident inhibitory responses to the CANS via modulation of the anterior right ganglionated plexus. Nonetheless, there are also cases with little to no effect on neuromodulation post-ablation. Purpose To assess the clinical and anatomical predictors of thermal ablation-induced CANS changes, as assessed via variations in resting HR post-ablation. Methods We retrospectively analyzed consecutive patients with symptomatic paroxysmal AF who underwent first-time PVI by cryoballoon (CB) or RF balloon (RFB) between January 2022 to November 2022. Patients who were not in sinus rhythm, had cardiac implantable electronic devices, or did not have a contrast-enhanced computed tomography before the ablation were excluded. Pre-ablation and 24-hour post-ablation ECGs in sinus rhythm were collected and analyzed to assess HR. Results A total of 83 patients (CB group, n = 43 vs RFB group, n = 40) were included in the analysis. Baseline patients’ characteristics were similar between the two groups (Table 1). A significant HR increase (ΔHR ≥ 15 bpm) at 24h post-ablation occurred in 31 patients [CB group 16 (52 %), RFB group 15 (48 %)]. Predictors of increased HR were the distance between superior vena cava (SVC) and RSPV (OR 0.51, CI 0.35 - 0.75, p value < 0.01) and age (OR 0.94, CI 0.89 - 0.99, p value < 0.01) (Table 2). Conclusions Single-shot thermal PVI influences the CANS, especially in patients with a shorter SVC - RSPV distance and in younger patients.Table 1Table 2

#1492 Metabolic switch in CD8+ T cells may influence immune response in chronic antibody-mediated rejection (CAMR)

Abstract Background and Aims CAMR is the main cause of chronic graft injury and subsequent graft loss, but its pathogenesis is still largely unclear. Our group demonstrated an increase in CD8+ T cells infiltrating biopsies from CAMR patients (PMID: 20595199), but their role remains to be defined. Recently, immunometabolism has emerged as a field connecting cell energy metabolism with the immune cell differentiation and functions. Particularly, the permanent aerobic glycolysis of CD8+ lymphocytes induces their transition from naïve to activated state and effector T cells (PMID: 37315709). Thus, the aim of the study was to investigate the molecular mechanisms underlying the development of CAMR by the analysis of gene expression profiles of CD8+ T lymphocytes in conjunction with their metabolic state. Method We enrolled 14 patients with biopsy-proven CAMR and 12 stable transplant recipients with normal graft histology and function (control group). CD8+ T cells were purified by CD8 MicroBeads (Miltenyi Biotec) then counted (TC20™ Automated Cell Counter). Gene expression profiles of CD8+ T lymphocytes isolated from both groups were assessed using Agilent microarrays. Data obtained were statistically and functionally evaluated (IPA software) and validated by qPCR. Oxygen consumption rate (OCR) in CD8+ and CD8- cells was measured by a Resipher oxygen sensing lid (Lucid Scientific Inc., USA). The contribution of mitochondrial respiration to OCR was investigated in isolated CD8+ T cells from CAMR and controls treated with rotenone/antimycin A (Rot/AA), oligomycin (Olygo) or carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to evaluate spare respiratory capacity (SRC), ATP-linked respiration, proton leak and maximal respiration. Results Gene expression profiles of CD8+ T cells showed a characteristic alteration of lipid metabolism in CAMR patients with the triglyceride biosynthesis pathway (p = 2.69E-3) as the most altered pathway and several genes de-regulated all potentially involved in the altered metabolic state of CD8+ T cells. Among others, we validated the increased expression of Nicotinamide Phosphoribosyltransferase (NAMPT) (FC = +2.39), the rate-limiting enzyme of nicotinamide adenine dinucleotide (NAD+) salvage pathway involved in the glycolysis progression, mostly activated in effector T cells. We then decided to evaluate the OCR and the metabolic state of CD8+T cells. Basal OCR was higher in CD8+ rather than CD8- cells, but with different trends among cells isolated from control and CAMR patients. Indeed, CD8+ cells from CAMR patients showed higher OCR compared to control, while OCR was lower in CD8- from CAMR rather than control patients. CD8+ OCR from both CAMR and control patients was mostly dependent on mitochondrial respiration, since it was deeply reduced by Rot/AA treatment. Interestingly, inhibitory response to Oligo treatment by CD8+ cells from CAMR patients was modest, while OCR was noticeably stimulated by FCCP in these cells, suggesting that mitochondrial oxygen uptake was not ATP linked but rather associated with increased uncoupling. These data suggest that CD8+ T cells from CAMR patients are activated effector T cells primarily utilizing aerobic glycolysis. To confirm this hypothesis, we evaluated the gene expression levels of: i) HIF1a, which involves permanent dependence on glycolysis even in the presence of oxygen (Warburg effect); ii) fatty acid transporter CD36, whose levels mimic an energy dependence on the beta oxidation of fatty acids, a process activated in memory T cells. We confirmed an increased expression of HIF1a and a reduced expression of CD36 in CD8+T cells from CAMR patients compared to controls (p<0.05), that confirm the T effector phenotype. Conclusion Our data suggest that CD8+ T cell from CAMR patients showed permanent dependency on glycolysis leading to altered immune phenotype and response, thus indicating novel opportunities to modulate the immune system for therapeutic purposes in kidney transplanted patients and reduce graft rejection.

Inhibition of reactive oxygen species production accompanying alternatively activated microglia by risperidone in a mouse ketamine model of schizophrenia.

Recent studies have highlighted the potential involvement of reactive oxygen species (ROS) and microglia, a major source of ROS, in the pathophysiology of schizophrenia. In our study, we explored how the second-generation antipsychotic risperidone (RIS) affects ROS regulation and microglial activation in the hippocampus using a mouse ketamine (KET) model of schizophrenia. KET administration resulted in schizophrenia-like behaviors in male C57BL/6J mice, such as impaired prepulse inhibition (PPI) of the acoustic startle response and hyper-locomotion. These behaviors were mitigated by RIS. We found that the gene expression level of an enzyme responsible for ROS production (Nox2), which is primarily associated with activated microglia, was lower in KET/RIS-treated mice than in KET-treated mice. Conversely, the levels of antioxidant enzymes (Ho-1 and Gclc) were higher in KET/RIS-treated mice. The microglial density in the hippocampus was increased in KET-treated mice, which was counteracted by RIS. Hierarchical cluster analysis revealed three morphological subtypes of microglia. In control mice, most microglia were resting-ramified (type I, 89.7%). KET administration shifted the microglial composition to moderately ramified (type II, 44.4%) and hyper-ramified (type III, 25.0%). In KET/RIS-treated mice, type II decreased to 32.0%, while type III increased to 34.0%. An invitro ROS assay showed that KET increased ROS production in dissociated hippocampal microglia, and this effect was mitigated by RIS. Furthermore, we discovered that a NOX2 inhibitor could counteract KET-induced behavioral deficits. These findings suggest that pharmacological inhibition of ROS production by RIS may play a crucial role in ameliorating schizophrenia-related symptoms. Moreover, modulating microglial activation to regulate ROS production has emerged as a novel avenue for developing innovative treatments for schizophrenia.

Pupil dilation reflects effortful action invigoration in overcoming aversive Pavlovian biases.

"Pavlovian" or "motivational" biases describe the phenomenon that the valence of prospective outcomes modulates action invigoration: Reward prospect invigorates action, whereas punishment prospect suppresses it. The adaptive role of these biases in decision-making is still unclear. One idea is that they constitute a fast-and-frugal decision strategy in situations characterized by high arousal, e.g., in presence of a predator, which demand a quick response. In this pre-registered study (N = 35), we tested whether such a situation-induced via subliminally presented angry versus neutral faces-leads to increased reliance on Pavlovian biases. We measured trial-by-trial arousal by tracking pupil diameter while participants performed an orthogonalized Motivational Go/NoGo Task. Pavlovian biases were present in responses, reaction times, and even gaze, with lower gaze dispersion under aversive cues reflecting "freezing of gaze." The subliminally presented faces did not affect responses, reaction times, or pupil diameter, suggesting that the arousal manipulation was ineffective. However, pupil dilations reflected facets of bias suppression, specifically the physical (but not cognitive) effort needed to overcome aversive inhibition: Particularly strong and sustained dilations occurred when participants managed to perform Go responses to aversive cues. Conversely, no such dilations occurred when they managed to inhibit responses to Win cues. These results suggest that pupil diameter does not reflect response conflict per se nor the inhibition of prepotent responses, but specifically effortful action invigoration as needed to overcome aversive inhibition. We discuss our results in the context of the "value of work" theory of striatal dopamine.

Acute effect of complexity in basketball on cognitive capacity

BackgroundExecutive functions, notably inhibition, significantly influence decision-making and behavioral regulation in team sports. However, more research must be conducted on individual player characteristics such as experience and motor skills. This study assessed how accumulated practical experience moderates inhibition in response to varying task difficulty levels.MethodsForty-four university students (age: 20.36 ± 3.13 years) participated in this study with two sessions: one followed standard 1 × 1 basketball rules (“Regular Practice”), while the other imposed motor, temporal, and spatial restrictions (“Restriction Practice”). Functional difficulty was controlled by grouping pairs with similar skill levels. Flanker and Go-Nogo tasks were used.ResultsIncreasing complexity worsened cognitive performance (inhibition). “Restriction Practice” showed a significantly slower and less accurate performance in both tests than “Regular Practice” (p < 0.001). Experience positively impacted test speed and accuracy (p < 0.001).ConclusionIn sports, acute cognitive impacts are intrinsically linked to the task’s complexity and the athlete’s cognitive resources. In this sense, it is essential to adjust individually the cognitive demands of the tasks, considering each athlete’s specific cognitive abilities and capacities.

Ginseng-DF ameliorates intestinal mucosal barrier injury and enhances immunity in immunosuppressed mice by regulating MAPK/NF-κB signaling pathways.

Dietary fiber (DF) has a good application prospect in effectively restoring the integrity of the intestinal mucosal barrier. Ginseng-DF has good physicochemical properties and physiological activity and shows positive effects in enhancing immunity. The aim of this study was to investigate the protective effect of Ginseng-DF on intestinal mucosal barrier injury induced by cyclophosphamide (CTX) in immunosuppressed mice and its possible mechanism. The effects of Gginseng-DF on immune function in mice were studied by delayed-type hypersensitivy, lymphocyte proliferation assay and NK cytotoxicity assay, the T lymphocyte differentiation and intestinal barrier integrity were analyzed by flow cytometry and western blot. Ginseng-DF (2.5% and 5%) could attenuate the inhibition of DTH response by CTX, promote the transformation and proliferation of lymphocytes, and stimulate NK effector cell activity. At the same time, Ginseng-DF could restore the proportion of CD4+/CD8+ T lymphocytes induced by CTX to different extents, improved spleen tissue damage, promoted the secretion of immunoglobulin IgG, and enhanced body immunity. More importantly, Ginseng-DF could up-regulate the contents of TNF-α, IFN-γ, IL-6 and IL-1β in serum and intestine of immunosuppressed mice to maintain the balance between Th1/Th2 cytokines, and improve the permeability of intestinal mucosal barrier. Meanwhile, Ginseng-DF could reduce intestinal epithelial cell apoptosis and improve intestinal adaptive immunity in CTX-induced immunosuppressed mice by regulating MAPK/NF-κB signaling pathway. Ginseng-DF can be used as a safe dietary supplement to enhance body immunity and reduce intestinal mucosal injury caused by CTX.

A rare cause of acute abdomen in a young immunocompetent woman

Streptococcus pyogenes is ahuman pathogenic, gram positive bacterium that primarily leads to pharyngitis or soft tissue infections. Primary peritonitis caused by S.pyogenes infection is rare and there are only afew published cases worldwide. Primary peritonitis due to other pathogens occurs in immunosuppressed conditions such as HIV or other chronic diseases. However, younger, healthy women are more likely to be affected by S.pyogenes peritonitis. At present, the underlying molecular mechanisms can only be speculated on. One possibility is that, similar to the clinical picture of streptococcal toxic shock syndrome (STSS), aspecific serotype of the Mprotein in combination with inhibition of the cell response of neutrophil granulocytes could play arole. In addition to peritonitis, the clinical picture may include other organ manifestations such as acute kidney damage or circulatory dysregulation. In terms of treatment, rapid pathogen-directed empirical antibiotic therapy is the treatment of choice. If there is no indication of secondary peritonitis, diagnostic laparoscopy can be dispensed with in the further diagnostic work-up.

Mass Spectrometry-Based Proteomics Analysis Unveils PTPRS Inhibits Proliferation and Inflammatory Response of Keratinocytes in Psoriasis.

In this study, we used data-independent acquisition-mass spectrometry (DIA-MS) to analyze the serum proteome in psoriasis vulgaris (PsO). The serum proteomes of seven healthy controls and eight patients with PsO were analyzed using DIA-MS. Weighted gene co-expression network analysis was used to identify differentially expressed proteins (DEPs) that were closely related to PsO. Hub proteins of PsO were also identified. The Proteomics Drug Atlas 2023 was used to predict candidate hub protein drugs. To confirm the expression of the candidate factor, protein tyrosine phosphatase receptor S (PTPRS), in psoriatic lesions and the psoriatic keratinocyte model, immunohistochemical staining, quantitative real-time polymerase chain reaction, and western blotting were performed. A total of 129 DEPs were found to be closely related to PsO. The hub proteins for PsO were PVRL1, FGFR1, PTPRS, CDH2, CDH1, MCAM, and THY1. Five candidate hub protein drugs were identified: encorafenib, leupeptin, fedratinib, UNC 0631, and SCH 530348. PTPRS was identified as a common pharmacological target for these five drugs. PTPRS knockdown in keratinocytes promoted the proliferation and expression of IL-1α, IL-1β, IL-23A, TNF-α, MMP9, CXCL8, and S100A9. PTPRS expression was decreased in PsO, and PTPRS negatively regulated PsO. PTPRS may be involved in PsO pathogenesis through the inhibition of keratinocyte proliferation and inflammatory responses and is a potential treatment target for PsO.

Resolvin D1 inhibits T follicular helper cell expansion in systemic lupus erythematosus

Objective Resolvin D1 (RvD1) is one of the specialized pro-resolving lipid mediators, which control inflammation resolution and regulate immune responses. Previous research showed that RvD1 could block the progression of systemic lupus erythematosus (SLE). However, the detailed mechanism remains to be fully understood. Method Plasma RvD1 levels, and proportions of T follicular helper cells (Tfh cells) were measured in SLE patients and healthy controls. Plasma RvD1 levels and proportions of Tfh cells were quantitated in an MRL/lpr mouse model of lupus treated with RvD1. Naïve CD4+ T cells were purified from MRL/lpr mice to study the effect of RvD1 on Tfh cell differentiation in vitro. Results In patients, there were significant negative correlations between plasma RvD1 levels and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) score, as well as between plasma RvD1 and anti-double-stranded DNA antibody levels, and numbers of peripheral Tfh cells and plasma cells. In MRL/lpr mice, the expected amelioration of disease phenotype and inflammatory response with RvD1 treatment correlated with decreased percentages of Tfh cells and plasma cells. In addition, the differentiation and proliferation of Tfh cells were markedly suppressed by RvD1 in vitro. Conclusion RvD1 may control SLE progression through the suppression of Tfh cell differentiation and subsequent inhibition of B-cell responses.

Enhancing genome editing in hPSCs through dual inhibition of DNA damage response and repair pathways

Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived hPSCs. Unlike Cas9-mediated knock-in, cytosine base editor and prime editor achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by cytosine base editor or prime editor additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of p53DD, a dominant negative p53, and three UNG inhibitor, engineered to specifically diminish base excision repair, improves cytosine base editor efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional prime editor system also significantly enhances prime editor efficiency in hPSCs. Thus, combined inhibition of the distinct cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.

Targeting the adenosinergic system in restless legs syndrome: A pilot, "proof-of-concept" placebo-controlled TMS-based protocol.

Restless Legs Syndrome (RLS) is a common sleep disorder characterized by an urge to move the legs that is responsive to movement (particularly during rest), periodic leg movements during sleep, and hyperarousal. Recent evidence suggests that the involvement of the adenosine system may establish a connection between dopamine and glutamate dysfunction in RLS. Transcranial magnetic stimulation (TMS) is a non-invasive electrophysiological technique widely applied to explore brain electrophysiology and neurochemistry under different experimental conditions. In this pilot study protocol, we aim to investigate the effects of dipyridamole (a well-known enhancer of adenosinergic transmission) and caffeine (an adenosine receptor antagonist) on measures of cortical excitation and inhibition in response to TMS in patients with primary RLS. Initially, we will assess cortical excitability using both single- and paired-pulse TMS in patients with RLS. Then, based on the measures obtained, we will explore the effects of dipyridamole and caffeine, in comparison to placebo, on various TMS parameters related to cortical excitation and inhibition. Finally, we will evaluate the psycho-cognitive performance of RLS patients to screen them for cognitive impairment and/or mood-behavioral dysfunction, thus aiming to correlate psycho-cognitive findings with TMS data. Overall, this study protocol will be the first to shed lights on the neurophysiological mechanisms of RLS involving the modulation of the adenosine system, thus potentially providing a foundation for innovative "pharmaco-TMS"-based treatments. The distinctive TMS profile observed in RLS holds indeed the potential utility for both diagnosis and treatment, as well as for patient monitoring. As such, it can be considered a target for both novel pharmacological (i.e., drug) and non-pharmacological (e.g., neuromodulatory), "TMS-guided", interventions.

Inhibition of LPS-Induced Skin Inflammatory Response and Barrier Damage via MAPK/NF-κB Signaling Pathway by Houttuynia cordata Thunb Fermentation Broth.

Houttuynia cordata Thunb is rich in active substances and has excellent antioxidant and anti-inflammatory activity. Scanning electron microscopy and gel permeation chromatography were used to analyze the molecular characteristics of the fermentation broth of Houttuynia cordata Thunb obtained through fermentation with Clavispora lusitaniae (HCT-f). The molecular weight of HCT-f was 2.64265 × 105 Da, and the polydispersity coefficient was 183.10, which were higher than that of unfermented broth of Houttuynia cordata Thunb (HCT). By investigating the active substance content and in vitro antioxidant activity of HCT-f and HCT, the results indicated that HCT-f had a higher active substance content and exhibited a superior scavenging effect on 2,2-diphenyl-1-picrylhydrazyl radicals and hydroxyl radicals, with IC50 values of 11.85% and 9.01%, respectively. Our results showed that HCT-f could effectively alleviate the increase in the secretion of inflammatory factors and apoptotic factors caused by lipopolysaccharide (LPS) stimulation, and had a certain effect on repairing skin barrier damage. HCT-f could exert an anti-inflammatory effect by down-regulating signaling in the MAPK/NF-κB pathway. The results of erythrocyte hemolysis and chicken embryo experiments showed that HCT-f had a high safety profile. Therefore, this study provides a theoretical basis for the application of HCT-f as an effective ingredient in food and cosmetics.

All‐Optically Triggered In‐Sensor Collision Detection and Warning Based on 2D Complementary Material Devices

AbstractPrecise and timely collision detection and warning are essential to ensure the safety of autonomous driving. However, existing collision detection systems based on image sensors and radars are prone to misjudgment in adverse environments such as darkness or rain. The lobula giant movement detector (LGMD) neuron found in locusts achieves potential collision detection in unpredictable environments without the need for object recognition algorithms. Existing artificial collision detectors inspired by LGMD suffer from complex device structures and sophisticated operating modes. Here, an LGMD‐inspired all‐optically triggered in‐sensor collision detector is presented by 2D complementary material devices (2D‐CMDs) composed of n‐type molybdenum disulfide (MoS2) and p‐type platinum diselenide (PtSe2) connected in series. The proposed 2D‐CMDs couple the positive photoconductivity of MoS2 and negative photoconductivity of PtSe2 in response to looming light, successfully mimicking the antagonism of excitatory and inhibitory responses in LGMD neurons to generate a nonmonotonic escape response. The 2D‐CMDs exhibit a simple device structure and all‐optically controlled operation mode, consuming only 1 nJ of energy for each collision detection. Furthermore, in‐sensor real‐time collision warning is realized by employing a Recurrent Neural Network (RNN) to predict alarm time based on the escape response of the proposed 2D‐CMDs.

In vitro analysis of interactions between Pseudomonas aeruginosa and Candida albicans treated with silver sulfadiazine in wound infections.

Microorganisms tend to rely on close relationships with other species to survive. Consequently, biofilms formed by interactions of different species have been shown to delay the wound healing process. Studies suggest these mixed-population infections contribute to the development of drug resistance and inhibition of host immune response. Silver sulfadiazine (SSD) has been shown to effectively decrease the risk of infection in an open wound. Typically, these are bacterial wound infections; however, the role of fungal species needs further attention. The purpose of this in vitro study was to determine the effect of SSD on interactions between Pseudomonas aeruginosa 09-009 (PA1) or P. aeruginosa 09-010 (PA2) and Candida albicans ATTC 64550 (CA). A mixture of 4 mL of tryptic soy broth (TSB) and 100 µL of CA and/or PA1 or PA2 (∼106 log cfu/mL) inoculums were deposited into either wells or vials. The wells or vials were then sonicated (50 W for 10 s) to separate microorganisms attached to the walls. After incubation, cell counts were performed at 24 and 48 h for each microorganism using specific media. Our results show that without SSD treatment, P. aeruginosa exhibits an inhibitory effect on C. albicans. Treatment with SSD demonstrated significant reduction of P. aeruginosa; however, C. albicans persisted. This experiment demonstrates that SSD was effective in reducing the bioburden of both P. aeruginosa strains after 24 and 48 h; however, it was not as effective in reducing C. albicans. The data suggest that for polymicrobial mixed infections containing Pseudomonas spp. and C. albicans, treatment with SSD may be beneficial but does not provide adequate microorganism eradication. As such, added treatments that provide coverage for Candida infection are necessary. Additional in vivo studies are needed to obtain a better understanding of the complex interactions between these organisms.

Multiple E3 ligases act as antiviral factors against SARS-CoV-2 via inducing the ubiquitination and degradation of ORF9b.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) open reading frame 9b (ORF9b) antagonizes the antiviral type I and III interferon (IFN) responses and is ubiquitinated and degraded via the ubiquitin-proteasome pathway. However, E3 ubiquitin ligases that mediate the polyubiquitination and degradation of ORF9b remain unknown. In this study, we identified 14 E3 ligases that specifically bind to SARS-CoV-2 ORF9b. Specifically, three E3 ligases, HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, induced K48-linked polyubiquitination and degradation of ORF9b, thereby attenuating ORF9b-mediated inhibition of the IFN response and SARS-CoV-2 replication. Moreover, each E3 ligase performed this function independent of the other two E3 ligases. Therefore, the three E3 ligases identified in this study as anti-SARS-CoV-2 host factors provide novel molecular insight into the virus-host interaction.IMPORTANCEUbiquitination is an important post-translational modification that regulates multiple biological processes, including viral replication. Identification of E3 ubiquitin ligases that target viral proteins for degradation can provide novel targets for antagonizing viral infections. Here, we identified multiple E3 ligases, including HECT, UBA, and WWE domain-containing E3 ubiquitin protein ligase 1 (HUWE1), ubiquitin protein ligase E3 component n-recognin 4 (UBR4), and UBR5, that ubiquitinated and induced the degradation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 9b (ORF9b), an interferon (IFN) antagonist, thereby enhancing IFN production and attenuating SARS-CoV-2 replication. Our study provides new possibilities for drug development targeting the interaction between E3 ligases and ORF9b.

Abstract PO4-23-08: Estrogen therapy induces receptor-dependent DNA damage enhanced by PARP inhibition in ER+ breast cancer

Abstract Although clinical evidence indicates that treatment with estrogens elicits anti-cancer effects in ~30% of patients with advanced endocrine-resistant ER+ breast cancer, the underlying mechanism of action is unclear and this treatment remains under-utilized. ER+ breast cancer cells with acquired resistance to long-term estrogen deprivation (LTED) were used to model resistance to aromatase inhibitors (AIs) seen clinically. Genome-wide CRISPR/Cas9 knockout screening and transcriptomic profiling of LTED cells highlighted DNA Damage Response as a required pathway for therapeutic response to the estrogen 17b-estradiol. Tumor specimens from 2 patients with advanced ER+ breast cancer showed increased DNA damage response upon treatment with 17b-estradiol therapy compared to baseline. LTED cells treated with 17b-estradiol exhibited replication-dependent markers of DNA damage and the DNA damage response prior to apoptosis, while parental cells did not. Such DNA damage was partially driven by the formation of DNA:RNA hybrids (R-loops), and RNase H1-mediated prevention of R-loop formation abrogated 17b-estradiol-induced DNA damage. Pharmacological suppression of the DNA damage response via poly(ADP-ribose) polymerase (PARP) inhibition with olaparib enhanced 17b-estradiol-induced DNA damage. PARP inhibition synergized with 17b-estradiol to suppress growth and prevent tumor recurrence in both BRCA1/2-mutant and BRCA1/2-wild-type cell line and patient-derived xenograft models. We therefore conclude that 17b-estradiol-induced ER activity drives DNA damage and growth inhibition in endocrine-resistant breast cancer cells, and inhibition of the DNA damage response using drugs such as PARP inhibitors can exacerbate transcriptional stress and enhance therapeutic response to 17b-estradiol. This concept is being explored clinically in the PHOEBE trial testing the combination of 17b-estradiol and olaparib (NCT05900895). These findings also warrant further study of other DNA damage response inhibitors in advanced ER+ breast cancer. Moreover, these data indicate that PARP inhibitors may have applications beyond homologous recombination-deficient tumors. Citation Format: Nicole Traphagen, Gary Schwartz, Steven Tau, Alyssa Roberts, Amanda Jiang, Sarah Hosford, Jonathan Marotti, Abigail Goen, Bianca Romo, Anneka Johnson, Emily Duffy, Eugene Demidenko, Paul Heverly, Yaron Mosesson, Shannon Soucy, Fred Kolling, Todd Miller. Estrogen therapy induces receptor-dependent DNA damage enhanced by PARP inhibition in ER+ breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-23-08.

Abstract PO4-28-11: Murine Breast Cancer Cell Culture Supernatant Induces CAF-like and TAM-like Traits in Normal Cells

Abstract Several studies in the past have demonstrated the role of cell-to-cell interaction (crosstalk) between tumor cells and normal (nontumorigenic) cells in cancer progression and metastasis. Within the tumor microenvironment (TME), these interactions have a potential to transform the phenotypes and the behaviors of normal cells. Application of the 2D in-vitro cultures has been limited due to its inability to replicate the complex in-vivo TME. Conditioned Medium (CM) obtained from cultured cancer cells contains secreted growth factors that potentially regulate the phenotype and the functionality of normal cells. In this study, a culture of normal murine fibroblast NIH3T3 and macrophage RAW 264.7 cells with conditioned medium (CM) obtained from malignant mammary epithelial 4T1 cells (4T1CM) resulted in an altered phenotype with increased cell viability. 4T1CM treated NIH3T3 and RAW 264.7, in comparison with the respective control cells, resulted in an upregulation of the genes including- α-smooth muscle actin (αSMA), IL-10, CD206, and vascular endothelial growth factor (VEGF). In addition, 4T1.CM treated NIH3T3 showed an EMT phenotype as indicated by the regulation of EMT markers such as, E-cadherin, β-catenin, N-cadherin, and Vimentin. Interestingly, an upregulation of cyclooxygenase-2 (COX-2) and programmed death-ligand 1 (PDL-1) was observed in 4T1CM treated RAW 264.7, a tendency towards exhibiting an inhibitory immune response. Intriguingly, NIH3T3 cells conditioned with 4T1CM demonstrated an upregulation of stemness markers including- sex determining region Y-box 2, and Aldehyde dehydrogenase. Collectively, our study suggested a role for 4T1CM in transforming the normal NIH3T3 and RAW 264.7 into cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). RNAseq experiments are underway to map differentially expressed genes (DEGs) that potentially regulate the 4T1CM induced transformation of NIH3T3 and RAW 264.7 cells. Furthermore, invitro drug testing in 3D model of these transformed cells to target the pathway intermediates may provide novel therapeutic intervention strategies. Citation Format: Biplov Sapkota, Naveen Chintalaramulu, Abhishek Pandit, Shilpa Thota, Rizwana Begum, Joseph Francis. Murine Breast Cancer Cell Culture Supernatant Induces CAF-like and TAM-like Traits in Normal Cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-11.

Interleukin-6 Modulates Action Potential Generation in Freshly Isolated Rat Dorsal Root Ganglia Neurons And its Stimulating Effect Is Exacerbated in Polycystic Kidney Disease

Our laboratory has demonstrated a central role for increased afferent nerve renal activity (ARNA) in the progression of cystogenesis in the PCK rat model of polycystic kidney disease (PKD). The underlying cause for the pathological increase in ARNA is unknown. Inflammatory cell infiltration and increased proinflammatory cytokine content, such as interleukin 6 (IL-6), have been reported in PKD. We hypothesized inflammatory cytokines directly increase sensory nerve activity, and this response is exacerbated in PKD. We measured action potential generation (AP) in freshly isolated dorsal root ganglion (DRG) neurons in PCK rats and non-cystic controls, and their response to IL-6. DRGs (T10-L1) were isolated from Sprague-Dawley (SD) or PCK rat (Age: 10-26 weeks) spinal cords, enzymatically digested and mechanically dissociated. Neurons were plated on coverslips, and AP were recorded within 24-hours (whole-cell patch-clamp). We tested the effects of IL-6 (0.04 μg/ml) and bradykinin (BK; 40μg/ml) on AP under 100, 200 pA, and 300 pA current clamp steps. IL-6 elicited either an excitatory response or inhibitory response in AP. In excitatory cells (EC), AP was normalized to BK peak response at +300 pA. In inhibited cells (IC), AP was normalized to baseline at +300 pA. PCK and IL-6 effects were assessed by two-way ANOVA (Bonferroni post hoc test;*p<.05); data as mean ± SEM. Proportion of ECs and ICs did not differ between SD and PCK rats. IL-6 increased AP at 300 pA from 0.48±0.28 (baseline) to 0.95±0.05 (p<.05) in SD ECs cells. BK further increased AP, showing no cell fatigue by IL-6 exposure. In PCK ECs, IL-6 increased AP at 100 pA (0.07±0.05 vs. 0.52±0.25), at 200 pA (0.47±0.27 vs. 1.39±0.81), and 300 pA (0.54±0.25 vs. 1.11±0.47). BK further increased AP at 100 pA to 0.70± 0.28 but decreased AP at 200 pA to 1.21±0.40 and at +300 pA to 1.00±0.00, indicating that neurons activity was likely plateaued by IL-6 at +200 and +300 pA. In both SD and PCK ICs, there was a main effect of IL-6 and BK treatment across all current steps (p<0.05). In SD ICs, IL-6 decreased AP at 200 pA (1.03±0.67 vs. 0.28±0.24) and 300 pA (1.00±0.00 vs. 0.28±0.24). BK had no effect at +200 pA and partially reversed the effect of IL-6 at +300 pA (AP increase to 0.53±0.27). In PCK ICs, IL-6 decreased AP at +200 pA (0.21±0.08 vs. 0.11±0.09) and +300 pA (1.00±0.00 vs. 0.42±0.24). BK increased AP at +200 pA (0.67±0.37) and +300 pA (0.71±0.33). These data partially support our hypothesis that IL-6 increases sensory nerve discharge. This effect was not observed uniformly across cells, as there were inhibitory and excitatory responses in both PCK and SD rats. We conclude that pro-inflammatory cytokine IL-6 can increase a subset of DRG neuron activity and responsiveness, an effect potentiated in PKD. This neuro-immune interaction might contribute to the underlying increase in ARNA and cystogenesis in PKD. Further experimentation is underway to test neuronal sensitivity to IL-6 and other inflammatory cytokines, as well as the posited potentiated effects in PKD. Supported in part by the PKD Foundation Research Award 1022534 (CB); NIDDK/PKD RRC Pilot and Feasibility Award U24DK126110 (CB). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Forelimb movements contribute to hindlimb cutaneous reflexes during locomotion in cats.

During quadrupedal locomotion, interactions between spinal and supraspinal circuits and somatosensory feedback coordinate forelimb and hindlimb movements. How this is achieved is not clear. To determine if forelimb movements modulate hindlimb cutaneous reflexes involved in responding to an external perturbation, we stimulated the superficial peroneal nerve in six intact cats during quadrupedal locomotion and during hindlimb-only locomotion (with forelimbs standing on stationary platform) and in two cats with a low spinal transection (T12-T13) during hindlimb-only locomotion. We compared cutaneous reflexes evoked in six ipsilateral and four contralateral hindlimb muscles. Results showed similar occurrence and phase-dependent modulation of short-latency inhibitory and excitatory responses during quadrupedal and hindlimb-only locomotion in intact cats. However, the depth of modulation was reduced in the ipsilateral semitendinosus during hindlimb-only locomotion. Additionally, longer-latency responses occurred less frequently in extensor muscles bilaterally during hindlimb-only locomotion while short-latency inhibitory and longer-latency excitatory responses occurred more frequently in the ipsilateral and contralateral sartorius anterior, respectively. After spinal transection, short-latency inhibitory and excitatory responses were similar to both intact conditions, while mid- or longer-excitatory responses were reduced or abolished. Our results in intact cats and the comparison with spinal-transected cats suggest that the absence of forelimb movements suppresses inputs from supraspinal structures and/or cervical cord that normally contribute to longer-latency reflex responses in hindlimb extensor muscles.