Ion Current Research Articles

Leukocyte Ig-like receptor B4 (Lilrb4a) alleviates cardiac dysfunction and isoproterenol-induced arrhythmogenic remodeling associated with cardiac fibrosis and inflammation

BackgroundHeart failure is usually accompanied by activation of the sympathetic nerve, and excessive activation of the sympathetic nerve promotes cardiac remodeling and cardiac dysfunction. In the isoproterenol (ISO)–induced animal model, it is often accompanied by myocardial hypertrophy, fibrosis, and inflammation. Leukocyte immunoglobulin-like receptor B4a (Lilrb4a), an immunosuppressive regulatory receptor, plays a vital role in cardiovascular disease. However, the effect of Lilrb4a on ventricular arrhythmia in an ISO-induced mouse model remains unclear. ObjectiveThe purpose of this study was to explore the role and molecular mechanism of Lilrb4a in ISO-induced arrhythmogenic remodeling. MethodsLilrb4a knockout mice and Lilrb4a overexpression mice were infused with ISO (15 mg/kg per 24 hours, 4 weeks). Echocardiography and histology evaluations of myocardial hypertrophy and cardiac structural remodeling were conducted. Surface electrocardiography and electrophysiologic examination were used to evaluate cardiac electrical remodeling and susceptibility to ventricular arrhythmias. Quantitative reverse transcriptase–polymerase chain reaction analysis and Western blotting were used to detect the expression levels of ion channel proteins and signal pathway proteins. ResultsThe results discovered that ISO induced cardiac hypertrophy, fibrosis, and inflammation and led to electrical remodeling and the occurrence of ventricular arrhythmias. Lilrb4a alleviated cardiac structural and electrical remodeling and protected against the occurrence of ventricular arrhythmias in ISO-induced mice by gain-of-function or loss-of-function approaches. The mechanism is that Lilrb4a inhibited NF-κB signaling and MAPK signaling activation mediated by transforming growth factor kinase 1. ConclusionLilrb4a alleviates cardiac dysfunction and ISO-induced arrhythmogenic remodeling associated with cardiac fibrosis and inflammation through the regulation of NF-κB signaling and MAPK signaling activation.

Pleiotropic physiological functions of Piezo1 in human body and its effect on malignant behavior of tumors.

Mechanosensitive ion channel protein 1 (Piezo1) is a large homotrimeric membrane protein. Piezo1 has various effects and plays an important and irreplaceable role in the maintenance of human life activities and homeostasis of the internal environment. In addition, recent studies have shown that Piezo1 plays a vital role in tumorigenesis, progression, malignancy and clinical prognosis. Piezo1 is involved in regulating the malignant behaviors of a variety of tumors, including cellular metabolic reprogramming, unlimited proliferation, inhibition of apoptosis, maintenance of stemness, angiogenesis, invasion and metastasis. Moreover, Piezo1 regulates tumor progression by affecting the recruitment, activation, and differentiation of multiple immune cells. Therefore, Piezo1 has excellent potential as an anti-tumor target. The article reviews the diverse physiological functions of Piezo1 in the human body and its major cellular pathways during disease development, and describes in detail the specific mechanisms by which Piezo1 affects the malignant behavior of tumors and its recent progress as a new target for tumor therapy, providing new perspectives for exploring more potential effects on physiological functions and its application in tumor therapy.

BmNPV p35 regulates apoptosis in Bombyx mori via a novel target of interaction with the BmVDAC2-BmRACK1 complex

Voltage-dependent anion channel 2 (VDAC2) is an important channel protein that plays a crucial role in the host response to viral infection. The receptor for activated C kinase 1 (RACK1) is also a key host factor involved in viral replication. Our previous research revealed that Bombyx mori VDAC2 (BmVDAC2) and B. mori RACK1 (BmRACK1) may interact with Bombyx mori nucleopolyhedrovirus (BmNPV), though the specific molecular mechanism remains unclear. In this study, the interaction between BmVDAC2 and BmRACK1 in the mitochondria was determined by various methods. We found that BmNPV p35 interacts directly with BmVDAC2 rather than BmRACK1. BmNPV infection significantly reduced the expression of BmVDAC2, and activated the mitochondrial apoptosis pathway. Overexpression of BmVDAC2 in BmN cells inhibited BmNPV-induced cytochrome c (cyto c) release, decrease in mitochondrial membrane potential as well as apoptosis. Additionally, the inhibition of cyto c release by BmVDAC2 requires the involvement of BmRACK1 and protein kinase C. Interestingly, overexpression of p35 inhibited cyto c release during mitochondrial apoptosis in a RACK1 and VDAC2-dependent manner. Even the mutant p35, which loses Caspase inhibitory activity, could still bind to VDAC2 and inhibit cyto c release. In summary, our results indicated that BmNPV p35 interacts with the VDAC2-RACK1 complex to regulate apoptosis by inhibiting cyto c release. These findings confirm the interaction between BmVDAC2 and BmRACK1, the interaction between p35 and the VDAC2-RACK1 complex, and a novel target that BmNPV p35 regulates apoptosis in Bombyx mori via interaction with the BmVDAC2-BmRACK1 complex. The result provide an initial exploration of the function of this interaction in the BmNPV-induced mitochondrial apoptosis pathway.

Dehydrated Biomimetic Membranes with Skinlike Structure and Function.

Novel vapor-permeable materials are sought after for applications in protective wear, energy generation, and water treatment. Current impermeable protective materials effectively block harmful agents but trap heat due to poor water vapor transfer. Here we present a new class of materials, vapor permeable dehydrated nanoporous biomimetic membranes (DBMs), based on channel proteins. This application for biomimetic membranes is unexpected as channel proteins and biomimetic membranes were assumed to be unstable under dry conditions. DBMs mimic human skin's structure to offer both high vapor transport and small molecule exclusion under dry conditions. DBMs feature highly organized pores resembling sweat pores in human skin, but at super high densities (>1012 pores/cm2). These DBMs achieved exceptional water vapor transport rates, surpassing commercial breathable fabrics by up to 6.2 times, despite containing >2 orders of magnitude smaller pores (1 nm vs >700 nm). These DBMs effectively excluded model biological agents and harmful chemicals both in liquid and vapor phases, again in contrast with the commercial breathable fabrics. Remarkably, while hydrated biomimetic membranes were highly permeable to liquid water, they exhibited higher water resistances after dehydration at values >38 times that of commercial breathable fabrics. Molecular dynamics simulations support our hypothesis that dehydration induced protein hydrophobicity increases which enhanced DBM performance. DBMs hold promise for various applications, including membrane distillation, dehumidification, and protective barriers for atmospheric water harvesting materials.

Correlation of source parameters and beam properties in the early operation of the full size ITER negative ion beam source

One of the requirements of Heating and current drive Neutral Beam injectors for ITER is a beam homogeneity greater than 90%, to achieve an optimal beam transmission while keeping the heat load consistently low on the acceleration electrodes. The large size and complexity of ITER negative ion source play a key role in determining the homogeneity of the negative ion current of each of the 1280 beamlets and their divergence, and it is studied in the full-scale prototype source SPIDER. In this work the plasma properties are studied by spectroscopic and electrostatic measurements in the drivers, where the plasma is generated, and in the expansion region, where the plasma drifts and negative ions are produced, and they are correlated with the properties of the beam. The non-homogeneous plasma density profile is related to the non-homogeneous availability of negative ions along the beam vertical profile, with and without cesium evaporation. Visible tomography, a technique capable of characterizing isolated beamlet properties, is used to study the beam’s dependence on plasma uniformity along the entire beam profile. Using these tools, it has been demonstrated how an increase in plasma density is linked to an improvement in beam homogeneity. The latter has been directly correlated with plasma homogeneity. The magnetic filter field and biases of the plasma grid and bias plate are responsible for the variation in plasma density and its homogeneity. Non-uniformities in the plasma’s top/bottom and left/right distributions have been studied and partially addressed experimentally. The first issue was resolved by adjusting the radio-frequency power supplied to the plasma in different vertical regions, while the second issue was addressed by reversing the direction of the magnetic filter field and increasing the plasma density.

Circular RNA-GRIN2B Suppresses Neuropathic Pain by Targeting the NF-κB/SLICK Pathway.

The role of circular RNAs (circRNAs) in neuropathic pain is linked to the fundamental physiological mechanisms involved. However, the exact function of circRNAs in the context of neuropathic pain is still not fully understood. The functional impact of circGRIN2B on the excitability of dorsal root ganglion (DRG) neurons was investigated using siRNA or overexpression technology in conjunction with fluorescence in situ hybridization and whole-cell patch-clamp technology. The therapeutic efficacy of circGRIN2B in treating neuropathic pain was confirmed by assessing the pain threshold in a chronic constrictive injury (CCI) model. The interaction between circGRIN2B and NF-κB was examined through RNA pulldown, RIP, and mass spectrometry assays. CircGRIN2B knockdown significantly affected the action potential discharge frequency and the sodium-dependent potassium current flux (SLICK) in DRG neurons. Furthermore, knockdown of circGRIN2B dramatically reduced the SLICK channel protein and mRNA expression in vivo and in vitro. Our research confirmed the interaction between circGRIN2B and NF-κB. These findings demonstrated that circGRIN2B promotes the transcription of the SLICK gene by binding to NF-κB. In CCI rat models, the overexpression of circGRIN2B has been shown to hinder the progression of neuropathic pain, particularly by reducing mechanical and thermal hyperalgesia. Additionally, this upregulation significantly diminished the levels of the inflammatory cytokines IL-1β, IL-6, and TNF-α in the DRG. Upon reviewing these findings, it was determined that circGRIN2B may mitigate the onset of neuropathic pain by modulating the NF-κB/SLICK pathway.

The E3 ubiquitin ligase ITCH negatively regulates intercellular communication via gap junctions by targeting connexin43 for lysosomal degradation

Intercellular communication via gap junctions has a fundamental role in regulating cell growth and tissue homeostasis, and its dysregulation may be involved in cancer development and radio- and chemotherapy resistance. Connexin43 (Cx43) is the most ubiquitously expressed gap junction channel protein in human tissues. Emerging evidence indicates that dysregulation of the sorting of Cx43 to lysosomes is important in mediating the loss of Cx43-based gap junctions in cancer cells. However, the molecular basis underlying this process is currently poorly understood. Here, we identified the E3 ubiquitin ligase ITCH as a novel regulator of intercellular communication via gap junctions. We demonstrate that ITCH promotes loss of gap junctions in cervical cancer cells, which is associated with increased degradation of Cx43 in lysosomes. The data further indicate that ITCH interacts with and regulates Cx43 ubiquitination and that the ITCH-induced loss of Cx43-based gap junctions requires its catalytic HECT (homologous to E6-AP C-terminus) domain. The data also suggest that the ability of ITCH to efficiently promote loss of Cx43-based gap junctions and degradation of Cx43 depends on a functional PY (PPXY) motif in the C-terminal tail of Cx43. Together, these data provide new insights into the molecular basis underlying the degradation of Cx43 and have implications for the understanding of how intercellular communication via gap junctions is lost during cancer development.

G6PD maintains the VSMC synthetic phenotype and accelerates vascular neointimal hyperplasia by inhibiting the VDAC1-Bax-mediated mitochondrial apoptosis pathway.

Glucose-6-phosphate dehydrogenase (G6PD) plays an important role in vascular smooth muscle cell (VSMC) phenotypic switching, which is an early pathogenic event in various vascular remodeling diseases (VRDs). However, the underlying mechanism is not fully understood. An IP‒LC‒MS/MS assay was conducted to identify new binding partners of G6PD involved in the regulation of VSMC phenotypic switching under platelet-derived growth factor-BB (PDGF-BB) stimulation. Co-IP, GST pull-down, and immunofluorescence colocalization were employed to clarify the interaction between G6PD and voltage-dependent anion-selective channel protein 1 (VDAC1). The molecular mechanisms involved were elucidated by examining the interaction between VDAC1 and apoptosis-related biomarkers, as well as the oligomerization state of VDAC1. The G6PD level was significantly elevated and positively correlated with the synthetic characteristics of VSMCs induced by PDGF-BB. We identified VDAC1 as a novel G6PD-interacting molecule essential for apoptosis. Specifically, the G6PD-NTD region was found to predominantly contribute to this interaction. G6PD promotes VSMC survival and accelerates vascular neointimal hyperplasia by inhibiting VSMC apoptosis. Mechanistically, G6PD interacts with VDAC1 upon stimulation with PDGF-BB. By competing with Bax for VDAC1 binding, G6PD reduces VDAC1 oligomerization and counteracts VDAC1-Bax-mediated apoptosis, thereby accelerating neointimal hyperplasia. Our study showed that the G6PD-VDAC1-Bax axis is a vital switch in VSMC apoptosis and is essential for VSMC phenotypic switching and neointimal hyperplasia, providing mechanistic insight into early VRDs.

Non-stoichiometric silicon nitride for future gravitational wave detectors

Silicon nitride thin films were deposited at room temperature employing a custom ion beam deposition (IBD) system. The stoichiometry of these films was tuned by controlling the nitrogen gas flow through the ion source and a process gas ring. A correlation is established between the process parameters, such as ion beam voltage and ion current, and the optical and mechanical properties of the films based on post-deposition heat treatment. The results show that with increasing heat treatment temperature, the mechanical loss of these materials as well as their optical absorption decreases producing films with an extinction coefficient as low as k=6.2(±0.5)×10−7 at 1064 nm for samples annealed at 900 ∘C. This presents the lowest value for IBD SiN x within the context of gravitational wave detector applications. The mechanical loss of the films was measured to be ϕ=2.1(±0.6)×10−4 once annealed post deposition to 900 ∘C.

Proteomic analysis of blood serum in bipolar disorder

IntroductionBipolar disorder (BD) often has symptoms similar to other mental disorders (BD), and there are no paraclinical criteria for differential diagnosis. (Geoffroy et al. Bip Dis 2017; 5 7). Published work on MD proteomics is scarce and focused on schizophrenia. (Dmitrieva et al. PeerJ. 2022; 10 e13907). Therefore, it is important to study potential biomarkers of BD using easily accessible material—blood serum (Rhee et al. Transl Psy 2023; 13 44). Identification of proteins involved in the pathogenesis of BD will help in the study of the pathogenetic mechanisms of BD, the development of differential diagnostic methods and pathogenetically based drugs.ObjectivesCarrying out a comparative proteomic analysis of blood serum from patients with BD and healthy individuals to identify potential biomarkersMethodsWe analyzed the protein spectrum of the blood serum of 14 patients with BD who were admitted during a depressive episode at the age of 32 [21;52] years with a disease duration of 8[5;11] years. The control group consisted of 10 mentally and somatically healthy individuals corresponding to the gender and age of the BD group. Blood serum was purified from 14 major proteins using affinity chromatography and separated by electrophoresis using the Laemmli method. After trypsinolysis, proteins were identified using HPLC/mass spectrometry on an Orbitrap instrument. Mass spectrometric analysis was performed on the Advanced Mass Spectrometry Core Facility of Skolkovo Institute of Science and Technology. Protein identification was carried out using the UniProtKB database using the Mascot search engine. The results were tested for significance using the nonparametric Fisher exact test with Yates correction.ResultsIn patients with BD, qualitative mass spectrometry revealed differential expression of 21 neurospecific proteins. Among them: Protein dispatched homolog 3, Ceroid-lipofuscinosis neuronal protein 6, SWI/SNF complex subunit SMARCC1, Neurogenic differentiation factor 4, Protein furry homolog-like, REST corepressor 1 – are involved in the proliferation, development and differentiation of neurons; Hemicentin-2, Dystrophin, Voltage-dependent L-type calcium channel subunit alpha-1D, Syntaxin-binding protein 5, Small conductance calcium-activated potassium channel protein 1– participate in synaptic transmission of ion transport and form receptors.ConclusionsStudying the role of these proteins in BD and their quantitative content in a larger number of patients is promising. This will help in the development of new diagnostic criteria and targets for drug therapy for BD.Support by the Russian Science Foundation grant No. 23-75-00023.Disclosure of InterestNone Declared

Narcissus cv. Hawera Leaves' Extract Cleaves DNA in Chlamydomonas reinhardtii by Inducing DSBs

The aim of this study is to go inside the mode of action of Narcissus cv. Hawera leaves' extract on Chlamydomonas reinhardtii by analyzing its chemical composition, genotoxic, mutagenic, and DNA damaging effects.
 GC/MS analysis of the methanol extract shows that alkaloids comprise about 23.93% of the total ion current (TIC). Considering the alkaloid pattern, lycorine is the major alkaloid (8.53% of TIC), followed by 6-epi-mesembrenol (4.59% of TIC), galanthamine (2.63% of TIC), and mesembrenone (2.01% of TIC). Other compounds in the extract are mono- and di-saccharides, organic acids, and fatty acids.
 Based on our experiments, both well-expressed genotoxic and DNA damaging potential of Narcissus cv. Hawera total leaves' extract was revealed for the first time. The most pronounced effect is obtained after the treatment with concentrations equal to or higher than 500 μg/ml. Cell survival decrease in a dose-dependent manner correlates with increased levels of induced DNA double-strand breaks (DSBs) but does not correspond with statistically confirmed mutagenic action. A good correlation is obtained between the levels of DSBs induced and cell survival rate.
 Here, for the first time, experimental evidence that Narcissus cv. Hawera leaves' extract can cleave DNA of Chlamydomonas reinhardtii inducing DSBs was reported by us. Our finding could be considered a good reason for further investigation of its biocidal capacity on other test systems for the purposes of “green” practice.

Self-powered sodium borohydride alkaline fuel cell for efficient removal of lead(II) and copper(II)

Heavy metals in industrial wastewater cause serious environmental pollution and they are easy to accumulate and difficult to degrade. Copper and lead are two typical heavy metals, and it is of great significance to remove them at the same time. This work shows a strategy of reducing and removing Pb(II) and Cu(II) by self-powered non-biological fuel cell (SP-nBFC) with dual-chamber reactor. Therein, sodium borohydride in alkaline solution as fuel (electron donor) was electrooxidized on nicke1 foam (NF) at anode and Pb(II) and Cu(II) ions (electron acceptors) in strong acidic solution were electro-reduced at Bi/MXene/GP cathode (bismuth nanoparticles and MXene modified graphite paper (GP)).Additionally, a proton exchange membrane, Nafion, was introduced into form complete circuit by offering ions channel. The results showed that Cu(II) and Pb(II) were reduced to Cu and Pb, and the SP-nBFC could produce electricity in this process without external energy consumption. The Bi/MXene/GP cathode could increase significantly removal efficiencies of Cu(II) and Pb(II), which were higher than about 37.0 %, 22.5 % for Cu(II) and 25.7 %, 16.3 % for Pb(II) than those of GP and MXene/GP cathode. This system could obtain a high Cu(II) removal efficiency (RE, 99.3 %) and moderate Pb(II) RE of 56.8 %, and acquire Cu and Pb sediments in cathode chamber. Furthermore, the proposed system could generate 923 mV of maximum potential and 1420 mW/m2 of maximum power density in 48 h. Thus, this SP-nBFC system provides a viable method for treatment of Cu(II) and Pb(II) pollution and generating electricity simultaneously, and also has prospective approach for other heavy metals remediation.

Transcriptome and open chromatin analysis reveals the process of myocardial cell development and key pathogenic target proteins in Long QT syndrome type 7

ObjectiveLong QT syndrome type 7 (Andersen–Tawil syndrome, ATS), which is caused by KCNJ2 gene mutation, often leads to ventricular arrhythmia, periodic paralysis and skeletal malformations. The development, differentiation and electrophysiological maturation of cardiomyocytes (CMs) changes promote the pathophysiology of Long QT syndrome type 7(LQT7). We aimed to specifically reproduce the ATS disease phenotype and study the pathogenic mechanism.Methods and resultsWe established a cardiac cell model derived from human induced pluripotent stem cells (hiPSCs) to the phenotypes and electrophysiological function, and the establishment of a human myocardial cell model that specifically reproduces the symptoms of ATS provides a reliable platform for exploring the mechanism of this disease or potential drugs. The spontaneous pulsation rate of myocardial cells in the mutation group was significantly lower than that in the repair CRISPR group, the action potential duration was prolonged, and the Kir2.1 current of the inward rectifier potassium ion channel was decreased, which is consistent with the clinical symptoms of ATS patients. Only ZNF528, a chromatin-accessible TF related to pathogenicity, was continuously regulated beginning from the cardiac mesodermal precursor cell stage (day 4), and continued to be expressed at low levels, which was identified by WGCNA method and verified with ATAC-seq data in the mutation group. Subsequently, it indicated that seven pathways were downregulated (all p < 0.05) by used single sample Gene Set Enrichment Analysis to evaluate the overall regulation of potassium-related pathways enriched in the transcriptome and proteome of late mature CMs. Among them, the three pathways (GO: 0008076, GO: 1990573 and GO: 0030007) containing the mutated gene KCNJ2 is involved that are related to the whole process by which a potassium ion enters the cell via the inward rectifier potassium channel to exert its effect were inhibited. The other four pathways are related to regulation of the potassium transmembrane pathway and sodium:potassium exchange ATPase (p < 0.05). ZNF528 small interfering (si)-RNA was applied to hiPSC-derived cardiomyocytes for CRISPR group to explore changes in potassium ion currents and growth and development related target protein levels that affect disease phenotype. Three consistently downregulated proteins (KCNJ2, CTTN and ATP1B1) associated with pathogenicity were verificated through correlation and intersection analysis.ConclusionThis study uncovers TFs and target proteins related to electrophysiology and developmental pathogenicity in ATS myocardial cells, obtaining novel targets for potential therapeutic candidate development that does not rely on gene editing.Graphical

Abstract 5904: Targeting NaV1.7 channels in pancreatic cancer: Implications for EF2K and P-Src/P-Fak/integrin β1 signaling pathways

Abstract Pancreatic cancer (PaCa) ranks as the fourth leading cause of cancer-related deaths in the United States and is among the top five worldwide. Its hallmark characteristics include early invasion, metastasis, resistance to chemotherapy and radiotherapy, and aggressive tumor progression. Evaluating new generation treatments with significant potential in this ongoing battle against cancer is crucial. Voltage-gated sodium channels (VGSC), long detected in various metastatic cell lines and tissue samples beyond breast, lung, and prostate cancer, are of particular interest. This study aimed to explore the therapeutic effects of inhibiting NaV1.7 and its neonatal variant, nNaV1.7, in pancreatic cancer cells. The study utilized human normal keratinocyte cell line HaCaT and pancreatic cancer cell lines PANC-1 and MiaPaCa-2. It investigated the roles and effects of adult and neonatal isoforms of the NaV1.7 channel in pancreatic cancer proliferation, invasion, and metastasis. The findings revealed significantly higher adult and neonatal NaV1.7 mRNA expressions in PANC-1 and MiaPaCa-2 cell lines compared to HaCaT cells. Specific siRNAs effectively downregulated channel mRNA and protein expressions. Notably, NaV1.7 siRNA treatments significantly inhibited cell proliferation and colony-forming capacity in PANC-1 and MiaPaCa-2 cells, without cytotoxic effects on HaCaT cells. Combining gemcitabine with specific NaV1.7 siRNAs enhanced drug efficacy in the PANC-1 cell line. Furthermore, NaV1.7 siRNAs significantly reduced cell invasion, migration, and wound healing in both cell lines. These siRNAs also induced apoptosis and G1 arrest in the cell cycle. They inhibited protein expressions involved in key signaling pathways, including P-Src/P-Fak/Integrin β1, EF2K, P-Akt, P-mTOR, PARP, Caspase-3/9, Cyclin D1/E1. This study is pioneering, as literature lacks research on the expression and function of NaV1.7 and nNaV1.7 in pancreatic cancer cells or tissue samples. In conclusion, NaV1.7 channel isoforms play a significant role in the development, progression, and metastasis of pancreatic cancer and present as a potential therapeutic target in PaCa. Citation Format: Mumin Alper Erdogan, Ayse Caner. Targeting NaV1.7 channels in pancreatic cancer: Implications for EF2K and P-Src/P-Fak/integrin β1 signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5904.

Kinetic modeling of ion formation during diesel combustion with different fuel injection timing

Ions are formed during the combustion process in internal combustion engines. The measurement of ions inside the combustion chamber produces reliable information about the combustion process. The present study focuses on the formation of ions inside the combustion chamber of diesel engines with different injection timing. For this purpose, a multi-zone thermodynamic model is utilized to simulate the closed cycle of the engine. To understand the kinetic behavior of the ions, the model is connected to an ionic chemical kinetics mechanism with 336 reactions and 81 species. Six important ionic reactions comprising 5 ions are used in the ionic mechanism. Dvode differential equation solver is also employed to calculate the energy and kinetics equations. The developed model has an acceptable accuracy in predicting the performance and pollutants of diesel engines. Based on the results, the ion formation is delayed by delaying the fuel injection timing. The maximum amount of in-cylinder ions depends on injection timing. In-cylinder ion current can predict the start of combustion accurately.

Abstract 2092: Genomic and proteomic analysis of glioblastoma recurrences during TTFields exposure

Abstract Glioblastoma (GB) is the most aggressive type of brain tumor, and current treatments are generally ineffective in preventing recurrence. Tumor Treating Fields (TTFields) are an innovative treatment that has been shown to improve patients' life expectancy in the last two decades. TTFields therapy has an antitumoral effect that involves different mechanisms and is an optional add-on to standard maintenance temozolomide. Although TTFields therapy initially slows tumor progression, it does not prevent tumor relapse in most patients. In the present investigation, we run a genomic and proteomic analysis of surgical material from 10 patients treated with TTFields. GB tissues were obtained from the primary tumor mass and from a second surgery after tumor relapse. All the patients, in addition to brain tumor standard therapies, were treated with TTFields for eighteen hours a day, the time span of treatment application was dependent on GB recurrence. Control samples were obtained from primary and secondary surgeries of 5 patients treated with standard therapy alone. The GB tissue extracts were analyzed with an RNAseq routine, and the results from the secondary surgery were compared with the data obtained from tumor specimens of the primary surgery. A common feature of transcripts among all the patients was the alterations of several pathways promoting the increase of intracellular oxidation and cell cycle regulation. In parallel with the analysis of patient specimens, we developed an in vitro GB cell relapse model. This was instrumental in investigating the beneficial contribution of TTFields action during tumor relapse, but also in uncovering the limitation of this therapeutic procedure. GB primary cultures obtained from patients' surgery were exposed for up to 12 days with 200 kHz TTFields stimulation using the inovitro™ system. After 12 days of continuous TTFields application, GB cells showed an average depolarization of the resting membrane potential, increasing oxidation, and acidification of the cytoplasm. Genomic analysis of treated cells compared with wild-type primary culture, denoted an increase of tumor stem cell markers, activation of several metabolic pathways, and upregulation of different ion channel protein transcripts. Our final goal is to identify specific molecular features enhanced by TTFields treatment to be used as a parallel therapy to fight GB recurrence. Citation Format: Francesca Cianci, Guido Rey, Dietmar Krex, Davide Ceresa, Paolo Malatesta, Michele Mazzanti. Genomic and proteomic analysis of glioblastoma recurrences during TTFields exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2092.

ENO1 contributes to the gemcitabine resistance of pancreatic cancer through the YAP1 signaling pathway.

Pancreatic cancer (PC), a leading cause of cancer-related deaths, has a 5-year survival rate of approximately 10%. α-Enolase (ENO1) is a junction channel protein involved in tumor cell apoptosis and chemoresistance. However, the role of ENO1 in PC remains unclear. The expression and prognosis of ENO1 levels were determined in PC using public databases based on The Cancer Genome Atlas (TCGA) data sets. Cell viability, half maximal inhibitory concentration (IC50), autophagy, apoptosis, and autophagy markers were examined using cell counting kit-8 (CCK-8), transmission electron microscope, flow cytometry assays, and immunoblot, respectively. Using the Gene Expression Omnibus (GEO) and TCGA data sets, we found that ENO1 was significantly enriched in PC tumor tissues, and high expression levels of ENO1 were associated with an unfavorable prognosis. Whereas ENO1 silencing suppressed proliferation, autophagy, and induced cell apoptosis in PC cells, and inhibited tumor growth in vivo. Mechanistically, knockdown of ENO1 enhanced cellular cytotoxicity of gemcitabine (GEM), as well as reducing the expression of yes-associated protein 1 (YAP1), a major downstream effector of the Hippo pathway in vitro. YAP1 promoted autophagy and protected PC cells from GEM-induced apoptotic cell death. Furthermore, YAP1 overexpression attenuated the inhibition effects of ENO1 silencing. Our results suggest that ENO1 overexpression promotes cell growth and tumor progression by increasing the expression of YAP1 in PC. Further studies are required to understand the detailed mechanisms between ENO1 and YAP1 in PC.

Inhibition the ubiquitination of ENaC and Na,K-ATPase with erythropoietin promotes alveolar fluid clearance in sepsis-induced acute respiratory distress syndrome

Sepsis-induced acute respiratory distress syndrome (ARDS) causes significant fatalities worldwide and lacks pharmacological intervention. Alveolar fluid clearance (AFC) plays a pivotal role in the remission of ARDS and is markedly impaired in the pathogenesis of ARDS. Here, we demonstrated that erythropoietin could effectively ameliorate lung injury manifestations and lethality, restore lung function and promote AFC in a rat model of lipopolysaccharide (LPS)-induced ARDS. Moreover, it was proven that EPO-induced restoration of AFC occurs through triggering the total protein expression of ENaC and Na,K-ATPase channels, enhancing their protein abundance in the membrane, and suppressing their ubiquitination for degeneration. Mechanistically, the data indicated the possible involvement of EPOR/JAK2/STAT3/SGK1/Nedd4–2 signaling in this process, and the pharmacological inhibition of the pathway markedly eliminated the stimulating effects of EPO on ENaC and Na,K-ATPase, and subsequently reversed the augmentation of AFC by EPO. Consistently, in vitro studies of alveolar epithelial cells paralleled with that EPO upregulated the expression of ENaC and Na,K-ATPase, and patch-clamp studies further demonstrated that EPO substantially strengthened sodium ion currents. Collectively, EPO could effectively promote AFC by improving ENaC and Na,K-ATPase protein expression and abundance in the membrane, dependent on inhibition of ENaC and Na,K-ATPase ubiquitination, and resulting in diminishing LPS-associated lung injuries.

Postbiotics of Lacticaseibacillus paracasei CECT 9610 and Lactiplantibacillus plantarum CECT 9608 attenuates store-operated calcium entry and FAK phosphorylation in colorectal cancer cells.

Store-operated Ca2+ entry (SOCE) is a major mechanism for Ca2+ influx in colorectal cancer (CRC) cells. This mechanism, regulated by the filling state of the intracellular Ca2+ stores, is mediated by the endoplasmic reticulum Ca2+ sensors of the stromal interaction molecules (STIM) family [stromal interaction molecule 1 (STIM1) and STIM2] and the Ca2+-release-activated Ca2+ channels constituted by Orai family members, with predominance of calcium release-activated calcium channel protein 1 (Orai1). CRC cells exhibit enhanced SOCE due to remodeling of the expression of the key SOCE molecular components. The enhanced SOCE supports a variety of cancer hallmarks. Here, we show that treatment of the colorectal adenocarcinoma cell lines HT-29 and Caco-2 with inanimate Lacticaseibacillus paracasei (CECT9610) and Lactiplantibacillus plantarum (CECT9608) attenuates SOCE, although no detectable effect is seen on SOCE in normal colon mucosa cells. The effect of Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics was mediated by downregulation of Orai1 and STIM1, while the expression levels of Orai3 and STIM2 remained unaltered. Treatment of HT-29 and Caco-2 cells with inanimate Lacticaseibacillus paracasei and Lactiplantibacillus plantarum impairs in vitro migration by a mechanism likely involving attenuation of focal adhesion kinase (FAK) tyrosine phosphorylation. Cell treatment with the Orai1 inhibitor synta-66 attenuates SOCE and prevents any further effect of Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics. Together, our results indicate for the first time that Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics selectively exert negative effects on Ca2+ influx through SOCE in colorectal adenocarcinoma cell lines, providing evidence for an attractive strategy against CRC.

Therapeutic effects of intermittent fasting on high-fat, high-fructose diet; involvement of jejunal aquaporin 1, 3, and 7

BackgroundAquaporins (AQPs) are transmembrane channel proteins. Aquaporin 1 (AQP1), Aquaporin 3 (AQP3), and Aquaporin 7 (AQP7) are expressed in the jejunum. The purpose of this study was to ascertain how a high-fat high-fructose diet (HFFD) and intermittent fasting (IF) affect AQP1, AQP3, and AQP7 expression in the rat jejunum. MethodsSixteen adult male rats were divided into control rats (n = 4) fed on a basal diet and water ad libitum for 12 weeks; IF control rats (n = 4) followed the IF protocol, HFFD-fed rats (n = 8) fed HFFD for eight weeks, and rats were randomized into two groups: HFFD only or HFFD and IF protocol from the beginning of the 9th week until the end of the experiment. The lipid profile values were assessed after 12 weeks. Jejunal oxidative markers (malondialdehyde and reduced glutathione) and AQP1, AQP3, and AQP7 mRNA expression were measured. Jejunal sections were used for morphometric analysis of villus length and crypt depth. Immunohistochemical evaluation of AQP1, AQP3, and AQP7 expression was also performed. ResultsIF ameliorates HFFD-induced lipid profile, oxidative stress, and jejunal morphometric changes. The results of both mRNA expression using PCR and immunohistochemistry showed a significant increase in AQP1, AQP3, and AQP7 expression in HFFD, whereas IF caused a decline in this expression. ConclusionThese findings suggest that IF can reduce inflammation, and oxidative stress and restore jejunal morphology caused by HFFD.