Signal Modification Research Articles

Quantitation of the PD-1/PD-L1 axis in non-small cell lung cancer by immuno-multiple reaction monitoring.

e14656 Background: Immuno-oncology revolutionized lung cancer treatment, but predictive biomarkers of response to checkpoint inhibitors (CPI) are still lacking. Only 50% of PD-L1 positive tumors by immunohistochemistry (IHC) respond to anti-PD-L1 treatment. Analytical variability and post-translational modifications (PTM) of the PD-1 signaling associated proteins (e.g.: glycosylation), can explain some of this discrepancy. Plus, the tumor immune microenvironment (TME) is complex and PD-L1 IHC alone is a flawed surrogate its status. Mass spectrometry based technologies have the potential to overcome these challenges by integrating sensitivity, specificity and absolute quantitation of proteins and PTMs in a standardized fashion. Here, we demonstrate the advantages of using anti-peptide antibodies to purify surrogate peptides followed by liquid-chromatography (LC) and multiple reaction monitoring (MRM), herby termed as iMRM, to gain new insight into the TME. Methods: To determine the concentration of PD-L1, PD-1, PD-L2, NT5E, LCK and ZAP70, we used unique and well detectable proteolytic peptides as surrogates. Our previously described protocol (ASCO 2022, #377181) allows robust quantitation of 13 peptides and monitors the glycosylation status of PD-L1, PD-L2, and PD-1. NSCLC samples were either fresh frozen (n = 42) or FFPE (n = 77) and sometimes both (n = 17). PD-L1 quantitation by iMRM was compared to PD-L1 IHC 22C3. Results: This multiplexed iMRM assay successfully quantified the PD-1/PD-L1 axis proteins in 96% of NSCLC patients (61/63). PD-L1 glycosylation levels ranged from 82 to 100% (n = 69, median = 100%, SD = 3.7%). Only PD-1 was significantly different between the fresh frozen (mean = 11±6 amol/µg total protein) and FFPE group (mean = 5±2 amol/µg total protein, ρ = 0.0001), all other peptides showed comparable levels. In our 17 matched FF/FFPE samples, PD-L1 moderately correlated (R = 0.45, ρ = 0.045) and the other peptides did not correlate (R < 0.03). Intra/intertumoral heterogeneity, differences in cellularity and tumor origin likely contributed to this discrepancy. IMRM results correlated moderately (R = 0.56, ρ < 0.01) with PD-L1 IHC. Most of these patients were not CPI-treated, but survival data was available. A trend was noted between the concentration of certain targets and patient survival. Linear regression was used to establish a cut-off for each peptide from which an immunoscore was calculated. The immunoscore could predict long-term survival (accuracy 75.4%) irrespective of tumor staging, grade or subtype and survival was significantly associated with the immunoscore (log-rank ρ = 0.005). Conclusions: Our iMRM workflow provides a new understanding of the TME in NSCLC through the PD-1/PD-L1 axis with an easy-to-read immunoscore. A set of 60 tumors from CPI-treated patients is currently being processed to validate the clinical utility of the assay in relation with CPI-response.

Very high-power short-duration pulmonary vein isolation combined with anterior chest position of dispersive electrodes

Abstract Funding Acknowledgements Type of funding sources: None. Introduction Pulmonary vein isolation (PVI) is a cornerstone management of atrial fibrillation (AF). Very high-power short-duration (vHPSD) radiofrequency catheter ablation (RFCA) can accelerate PVI procedure. Recently, special attention has been paid to the position of dispersive electrodes (DE) for optimization of RFCA application. However, these two methods have not yet been combined in the setting of PVI. Purpose To determine feasibility, safety, efficacy, and overall performance of vHPSD PVI with anterior chest position of DE. Methods Consecutive patients undergoing vHPSD PVI using flexible-tip catheter and anterior position of DE were included in the study. vHPSD protocol consisted of 70W RFCA applications lasting 4s. If needed, RFCA applications were extended using 50W until R-positive unipolar signal modification and/or until impedance drop ≥10% from baseline was achieved or were terminated in case of steam-pop occurrence. Demographical, clinical, procedural and follow-up data were collected. Results A total number of 43 patients (25 males, age 61±14years) were included in the study. 34 patients had paroxysmal AF (parox-AF group) and 9 patients had persistent AF (pers-AF group). Mean procedure time was 61±7min, mean RFCA time was 685±204s. All PVs were successfully isolated. Audible steam pops without sequele occurred in 6 (14%) cases. Major complications included one TIA without sequele. During follow-up 33 (77%) patients remained AF-free: 26 (76%) in parox-AF group vs 7 (78%) in pers-AF group (log rank p=0.0166). No atrioesophageal fistula or evidence of esophageal injury occurred. Conclusions Anterior chest position of DE combined with vHPSD approach for PVI is feasible, appears safe and effective. More data on possible protective properties against atrioesophageal fistula occurrence using the presented approach is warranted.

Anti-human-TIGIT agonistic antibody ameliorates autoimmune diseases by inhibiting Tfh and Tph cells and enhancing Treg cells

T cells play important roles in autoimmune diseases, but it remains unclear how to optimally manipulate them. We focused on the T cell immunoreceptor with Ig and ITIM domains (TIGIT), a coinhibitory molecule that regulates and is expressed in T cells. In autoimmune diseases, the association between TIGIT-expressing cells and pathogenesis and the function of human-TIGIT (hu-TIGIT) signalling modification have not been fully elucidated. Here we generated anti-hu-TIGIT agonistic monoclonal antibodies (mAbs) and generated hu-TIGIT knock-in mice to accurately evaluate the efficacy of mAb function. Our mAb suppressed the activation of CD4+ T cells, especially follicular helper T and peripheral helper T cells that highly expressed TIGIT, and enhanced the suppressive function of naïve regulatory T cells. These results indicate that our mAb has advantages in restoring the imbalance of T cells that are activated in autoimmune diseases and suggest potential clinical applications for anti-hu-TIGIT agonistic mAbs as therapeutic agents.

Data-Driven Automatic Generation Control of Interconnected Power Grids Subject to Deception Attacks

In this paper, a Data-Driven Adaptive Control (DDAC) technique is proposed for the Automatic Generation Control (AGC) problem of an interconnected power grid subject to Deception Attack (DA). The emergence of the Internet of Things (IoT) and the advancement of communication technologies have provided an opportunity for power system operators and designers to compensate for the lack of an appropriate model using a huge amount of data. However, they have also caused security challenges in the grid due to malicious attackers. This paper focuses on the attack to the control network which carries the AGC signals between the secondary and local primary frequency controllers. Intentional modifications of AGC signals during an attack may result in frequency instability because of saturation in governor signals. To counteract such an attack, a DDAC is suggested for a multi-area power system in which, the system model is dynamically updated using real-time input and output signals. The model includes the attacker’s behavior, thus empowering the control system to act against it. The stability of the proposed controller is proved using the Lyapunov stability theory when the DA causes input saturation. Simulation results show that it can successfully tolerate a class of deception attacks and keep the multi-area power grid stable.

A cellular platform for the evaluation of the effect of O-glycan modification on B cell receptor signaling

Abstract The importance of N-glycosylation, a ubiquitous post-translational modification (PTM) of cell-surface and secreted proteins in vertebrates, in the regulation of B cell receptor (BCR) signaling has been well documented. On the other hand, the importance of O-glycosylation, another type of ubiquitous PTM, in B cell function is getting recognized only recently. A simplified system to study the effects of O-glycan modifications in BCR signaling would facilitate the efforts to understand the mechanism of B cell regulation by O-glycans. To this end, we reconstituted BCR components and CD45 with orthogonal self-labeling protein tags in the mouse myeloma J558L cell line, which is deficient in the components of BCR and several co-receptors. The enzymes that modify O-glycans were further introduced, and the cells were subjected to imaging-based quantitative analysis of the distance between BCR and CD45, as well as the evaluation of BCR signaling by Western blotting. Gene expression profiles among the cells were also compared by RNA sequencing. Our study revealed that core 2 branching of O-glycans has minimal effect on the distance between BCR and CD45, whereas the addition of an α2–8-linked sialic acid by ST8Sia-VI reduces the distance between them, facilitating the downstream BCR signaling. Our system may be adapted for the functional evaluation of other PTMs in BCR signaling. Supported by a grant from the Ministry of Science and Technology (MOST)/National Science and Technology Council (NSTC) of Taiwan (MOST 109-2311-B-001-009-MY3)

PO-01-200 UNIPOLAR SIGNAL MODIFICATION-GUIDED RADIOFREQUENCY ABLATION

Positive atrial unipolar signal modification (USM) during catheter ablation has been shown to reflect irreversible transmural lesions. To assess the relationship between tissue thickness, power modification, and lesion transmurality in radiofrequency ablation (RFA) guided by USM, and to compare the ablation time between ablation index (AI) guided RFA and USM-guided RFA. In-vivo: RFA single lesions guided by USM were created in the right and left atrium of Yorkshire swine using 30W-35W power. Lesions were assessed for depth, width, and transmurality. Secondary analysis stratifying for tissue thickness (thickness <3mm and thickness ≥3mm) was performed. In-human: in 5 consecutive patients undergoing RFA for paroxysmal atrial fibrillation guided by AI using 35-40W power, the time to USM was recorded and was compared with the real time needed for AI guided ablation (AI=380-550). USM were obtained using the PureEP System from BioSig Technologies for signal recordings. Unipolar ablation signals were collected and compared at high pass filter settings of 0.05Hz, 0.5Hz, 1.0Hz, and 5Hz. In-vivo: A total of 13 swine were used (weight 83±6kg) with a total of 50 ablations. Using 30-35W (n=50 lesions, contact-force =12±3g), time to USM was 6±2.2 seconds, impedance drop was 18±8 Ohm, and 90% of the lesions were transmural. Lesion dimensions were (shown in mean ± std in mm): tissue thickness 2±1.1mm, depth 1.95±0.6mm, and width 9.2±0.6mm. The average AI at USM in swine was 280±48. Secondary analysis demonstrated that in lesions with tissue-thickness <3mm (n=38; 1.5mm-2.5mm) transmurality was 100% for USM guided ablations, whereas in lesions with tissue-thickness ≥3mm (n=12; 3mm-5.5mm) transmurality was achieved only in 33% of the cases when guided by USM. In-humans: A total of 340 lesions were performed using 35-40W power. Mean ablation time (real time for AI guided ablation) was 24±9.5 seconds, whereas the USM were noted at a mean time of 8±3 seconds (delta of 18±11 seconds; p<0.001 for the comparison; Figure). For atrial tissue thickness <3mm, USM-guided ablation successfully resulted in transmural lesions. The time needed for ablation was very short and was less than one third of the time needed for AI guided ablations. Further studies are needed to find the optimal ablation settings for tissue thickness ≥3mm where current methods of using AI guided ablation might be too prolonged.

Mitophagy as a mitochondrial quality control mechanism in myocardial ischemic stress: from bench to bedside.

Myocardial ischemia reduces the supply of oxygen and nutrients to cardiomyocytes, leading to an energetic crisis or cell death. Mitochondrial dysfunction is a decisive contributor to the reception, transmission, and modification of cardiac ischemic signals. Cells with damaged mitochondria exhibit impaired mitochondrial metabolism and increased vulnerability to death stimuli due to disrupted mitochondrial respiration, reactive oxygen species overproduction, mitochondrial calcium overload, and mitochondrial genomic damage. Various intracellular and extracellular stress signaling pathways converge on mitochondria, so dysfunctional mitochondria tend to convert from energetic hubs to apoptotic centers. To interrupt the stress signal transduction resulting from lethal mitochondrial damage, cells can activate mitophagy (mitochondria-specific autophagy), which selectively eliminates dysfunctional mitochondria to preserve mitochondrial quality control. Different pharmacological and non-pharmacological strategies have been designed to augment the protective properties of mitophagy and have been validated in basic animal experiments and pre-clinical human trials. In this review, we describe the process of mitophagy in cardiomyocytes under ischemic stress, along with its regulatory mechanisms and downstream effects. Then, we discuss promising therapeutic approaches to preserve mitochondrial homeostasis and protect the myocardium against ischemic damage by inducing mitophagy.

In silico interrogation of the miRNAome of infected hematopoietic cells to predict processes important for human cytomegalovirus latent infection

Human cytomegalovirus (HCMV) latency in CD34+ progenitor cells is the outcome of a complex and continued interaction of virus and host that is initiated during very early stages of infection and reflects pro- and anti-viral activity. We hypothesized that a key event during early infection could involve changes to host miRNAs, allowing for rapid modulation of the host proteome. Here, we identify 72 significantly upregulated miRNAs and three that were downregulated by 6hpi of infection of CD34+ cells which were then subject to multiple in silico analyses to identify potential genes and pathways important for viral infection. The analyses focused on the upregulated miRNAs and were used to predict potential gene hubs or common mRNA targets of multiple miRNAs. Constitutive deletion of one target, the transcriptional regulator JDP2, resulted in a defect in latent infection of myeloid cells; interestingly, transient knockdown in differentiated dendritic cells resulted in increased viral lytic IE gene expression, arguing for subtle differences in the role of JDP2 during latency establishment and reactivation of HCMV. Finally, in silico predictions identified clusters of genes with related functions (such as calcium signaling, ubiquitination, and chromatin modification), suggesting potential importance in latency and reactivation. Consistent with this hypothesis, we demonstrate that viral IE gene expression is sensitive to calcium channel inhibition in reactivating dendritic cells. In conclusion, we demonstrate HCMV alters the miRNAome rapidly upon infection and that in silico interrogation of these changes reveals new insight into mechanisms controlling viral gene expression during HCMV latency and, intriguingly, reactivation.

Detecting memory content in firing rate signals using a machine learning approach: A fractal analysis

While top-down modulation can be observed by increases in neuronal firing in some brain areas, modulation in other areas demonstrates complex nonlinear dynamics. When these signal modifications cannot be identified by classical methods, fractal analysis can be used to better describe the resulting changes. It was previously shown that the Higuchi dimension could detect such modification. However, these changes likely extend to multiple fractal dimensions. In this paper, machine learning algorithms were employed to extract the fractal features of neuronal firing and detect the occurrence of top-down modulation. We assessed the performance of classifiers trained on different fractal features as well as the Fourier transform and wavelet transform-based features obtained from the firing rate signals during a spatial memory task to measure the ability of individual neurons in the middle temporal cortex to hold memory information. By comparing the results of the different classifiers, Higuchi fractal dimension was confirmed as the strongest indicator of working memory content in cortical firing patterns compared to other fractal features. We demonstrated the utility of this approach and observed that the middle temporal cortex likely encodes spatial working memory based on linear and nonlinear temporal features rather than simply increasing the average firing rate.

Generation of an A549 ISRE-luciferase stable cell line

With its human lung origin, A549 cell line is a designated cellular model for viral respiratory infections studies. As such infections are known to lead to innate immune responses, various IFN signaling modifications occur in infected cells and have to be considered in respiratory viruses experiments. Here, we describe the generation of an A549 stable cell line that expresses firefly luciferase upon interferon-β stimulation, as well as upon RIG-I transfection and upon influenza A virus infection. Of the 18 clones generated, the first one, namely A549-RING1, demonstrated appropriate luciferase expression in the different conditions tested. This newly established cell line may therefore be used to decipher the impact of viral respiratory infection on innate immune response depending on IFN stimulation, without any plasmid transfection step. A549-RING1 can be provided upon request.

Abstract 2627: The small molecule KRAS inhibitor, TEB-17231, blocks tumor progression and overcomes KRASG12C inhibitor mediated resistance

Abstract KRAS pathways are frequently upregulated in cancers bearing KRAS mutations or by other signaling modifications. Also, resistance to KRASG12C inhibitors and other KRAS/RAF/MEK pathway drugs is commonly observed in the treated cancer patients. A vital unmet need is the development of agents that potently and safely inhibit the altered KRAS signaling in these settings. A broadly acting KRAS mutation small molecule inhibitor lead series was developed using KRAS domain structure-guided scaffold modifications starting from a group of KRASG12C inhibitors. Optimized properties of these compounds were evaluated by tumor cell selectivity in vitro and pharmacophore elaboration. A novel lead series was identified with robust tumor cell growth inhibition, exemplified with the TEB-17112 lead candidate, having IC50 of inhibition against multiple KRAS-altered cell lines: NCI-H358 (KRAS-G12C, 33.6nM), AGS (KRAS-G12D, 23.5 nM), SW480 (KRAS-G12V, 22.5 nM) and A375 (KRAS-wildtype, 58.9 nM) respectively. TEB-17112 demonstrated inhibition of SW480 and H358 mouse xenografts at 25 or 50 mg/kg PO QD (21 days) without significant body weight loss. On further optimization of the lead series, TEB-17231 had on average a higher potency against KRAS mutant tumor cell lines, including H358 (12 nM), AGS (5.4 nM), SW480 (3.6 nM), and A427 (KRAS-G12D, 9.7 nM). With a candidate group of 7 compounds from this lead series, there was consistently a 5- to 6-fold selectivity of inhibition for KRAS mutant over KRAS wildtype cell lines. The lead candidate TEB-17231 was also observed to inhibit cell lines with other KRAS mutations: NCI-H1734 (KRAS-G13C, 6.5 nM), MDA-MB-231 (KRAS-G13D,11.2 nM), RPMI-8226 (KRAS-G12A, 11.3 nM), Calu-6 (KRAS-Q61K,19.8 nM), KP-2 (KRAS-G12R, 24.1 nM), LS123 (KRAS-G12S, 21 nM), as well as HRAS and NRAS mutant cell lines. TEB-17231 inhibits phospho-ERK downstream signaling in several tumor cell lines. Further, TEB-17231 exhibited tumor growth inhibition (IC50) in KRAS-G12C Ba/F3 (14 nM) and derivative cell lines that acquired resistance to G12C inhibitor MRTX849 by the additional KRAS domain mutations as follows: H95Q (32 nM), H95D (40 nM), Y96C (39 nM), Y96D (20 nM) and R68S (22 nM), compared with greater than 1μM for MRTX849. TEB-17231 induces late apoptosis of NCI-H358 cells in a manner that is time and concentration dependent. In addition to the improved selectivity and potency, TEB-17231 demonstrated satisfactory pharmacokinetics and oral bioavailability in mice (55%). Furthermore, with the SW480 (G12V) in vivo xenograft model, TEB-17231 showed clear tumor growth inhibition with QD dosing for 4-8 mg/kg PO without body weight loss. These properties highlight TEB-17231 as a potential clinically actionable inhibitor for the KRAS-modified tumor population and warrant investigation of TEB-17231 in combination with other agents in the future. Citation Format: Zusheng Xu, David T. Weaver, Robert Drakas, Yangtong Lou. The small molecule KRAS inhibitor, TEB-17231, blocks tumor progression and overcomes KRASG12C inhibitor mediated resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2627.

Abstract 94: Advanced preclinical modeling reveals unique monosomy 3 associated biology in late-stage uveal melanoma

Abstract Uveal melanoma (UM) is a rare and lethal malignancy with very limited therapeutic options characterized by mutually exclusive activating mutations in GNAQ and GNA11, resulting in overactive proliferative signaling of Gαq/11 heterotrimeric G proteins. Additionally, tumors typically present with mutually exclusive mutations in one of three proteins with little functional overlap: BAP1, SF3B1, or EIF1AX. Metastatic progression of late-stage uveal melanoma is highly associated with chromosome 3 copy-loss, a unique chromosomal event (monosomy 3 or M3). However, the underlying biology of this distinct copy-loss event, and whether this event directly contributes towards the metastatic phenotype observed in late-stage disease, has largely been unexplored partially due to a lack of available models in the field. Here, using CRISPR-based centromere targeting we artificially created multiple isogenic clones exhibiting monosomy 3 (M3) from a well characterized disomy 3 (D3) UM cell model. Remarkably, the selective loss of one copy of chromosome 3 was sufficient to recapitulate clinical hallmarks of metastatic UM, including pigmentation loss, alterations in cell morphology and expression of late-stage disease markers. In addition, we also show how chromosome 3 copy-loss increases invasive potential and site-specific liver metastasis upon orthotopic implantation. Furthermore, successful development of this isogenic system has allowed for a comprehensive understanding of the molecular signaling, transcriptomic changes and chromatin-level modifications directly regulated by chromosome 3 copy-loss. Specifically, the monosomy 3 event emerged as the major contributor towards the molecular variance across D3 vs. M3 UM contexts and the primary driving force underpinning the large-scale epigenomic rewiring observed in M3 isogenic clones via transposase-accessible chromatin with sequencing (ATACseq). These observations together supported a scenario where the epigenomic rewiring associated to a selective genomic alteration, in this case the loss of one copy of chromosome 3, directs the acquisition of a new and aggressive phenotype. Current studies are focused on defining how these epigenomic alterations contribute towards the metastatic phenotype and identifying context-specific vulnerabilities associated with chromosome 3 copy-loss to develop novel therapeutics strategies to treat late-stage metastatic disease. Citation Format: Johnathon L. Rose, Sanjana Srinivasan, Meng He, Rosalba Minelli, Chiu-Yi Liu, Jason Gay, Joseph R. Daniele, Michael Peoples, Melinda Soeung, Vandhana Ramamoorthy, Anastasia Lopez, Charles Deckard, Brooke Meyers, Edoardo Del Poggetto, Daniel Zamler, Justin Huang, Luigi Perelli, Khalida Wani, Christopher Bristow, Ningping Feng, Christopher P. Vellano, Joe Marszalek, Alexander J. Lazar, Andrew Futreal, Giulio F. Draetta, Scott E. Woodman, Timothy Heffernan, Giannicola Genovese, Alessandro Carugo, Virginia Giuliani. Advanced preclinical modeling reveals unique monosomy 3 associated biology in late-stage uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 94.

Elm tree defences against a specialist herbivore are moderately primed by an infestation in the previous season.

The studies of the long-term effects of insect infestations on plant anti-herbivore defences tend to focus on feeding-induced damage. Infestations by an entire insect generation, including egg depositions as well as the feeding insects, are often neglected. Whilst there is increasing evidence that the presence of insect eggs can intensify plants' anti-herbivore defences against hatching larvae in the short term, little is known about how insect infestations, including insect egg depositions, affect plant defences in the long term. We addressed this knowledge gap by investigating long-term effects of insect infestation on elm's (Ulmus minor Mill. cv. 'Dahlem') defences against subsequent infestation. In greenhouse experiments, elms were exposed to elm leaf beetle (ELB, Xanthogaleruca luteola) infestation (adults, eggs and larvae). Thereafter, the trees cast their leaves under simulated winter conditions and were re-infested with ELB after the regrowth of their leaves under simulated summer conditions. Elm leaf beetles performed moderately worse on previously infested elms with respect to several developmental parameters. The concentrations of the phenylpropanoids kaempferol and quercetin, which are involved in egg-mediated, short-term effects on elm defences, were slightly higher in the ELB-challenged leaves of previously infested trees than in the challenged leaves of naïve trees. The expression of several genes involved in the phenylpropanoid pathway, jasmonic acid signalling, and DNA and histone modifications appeared to be affected by ELB infestation; however, prior infestation did not alter the expression intensities of these genes. The concentrations of several phytohormones were similarly affected in the currently challenged leaves of previously infested trees and naïve trees. Our study shows that prior infestation of elms by a specialised insect leads to moderately improved defences against subsequent infestation in the following growing season. Prior infestation adds a long-term effect to the short-term enhancer effect that plants show in response to egg depositions when defending against hatching larvae.

Neutrophil Extracellular Traps and Cancer: Trapping Our Attention with Their Involvement in Ovarian Cancer.

Neutrophils, the most abundant circulating leukocytes, play a well-known role in defense against pathogens through phagocytosis and degranulation. However, a new mechanism involving the release of neutrophil extracellular traps (NETs) composed of DNA, histones, calprotectin, myeloperoxidase, and elastase, among others, has been described. The so-called NETosis process can occur through three different mechanisms: suicidal, vital, and mitochondrial NETosis. Apart from their role in immune defense, neutrophils and NETs have been involved in physiopathological conditions, highlighting immunothrombosis and cancer. Notably, neutrophils can either promote or inhibit tumor growth in the tumor microenvironment depending on cytokine signaling and epigenetic modifications. Several neutrophils' pro-tumor strategies involving NETs have been documented, including pre-metastatic niche formation, increased survival, inhibition of the immune response, and resistance to oncologic therapies. In this review, we focus on ovarian cancer (OC), which remains the second most incidental but the most lethal gynecologic malignancy, partly due to the presence of metastasis, often omental, at diagnosis and the resistance to treatment. We deepen the state-of-the-art on the participation of NETs in OC metastasis establishment and progression and their involvement in resistance to chemo-, immuno-, and radiotherapies. Finally, we review the current literature on NETs in OC as diagnostic and/or prognostic markers, and their contribution to disease progression at early and advanced stages. The panoramic view provided in this article might pave the way for enhanced diagnostic and therapeutic strategies to improve the prognosis of cancer patients and, specifically, OC patients.

Towards Probing Conformational States of Y2 Receptor Using Hyperpolarized 129Xe NMR.

G protein-coupled receptors can adopt many different conformational states, each of them exhibiting different restraints towards downstream signaling pathways. One promising strategy to identify and quantify this conformational landscape is to introduce a cysteine at a receptor site sensitive to different states and label this cysteine with a probe for detection. Here, the application of NMR of hyperpolarized 129Xe for the detection of the conformational states of human neuropeptide Y2 receptor is introduced. The xenon trapping cage molecule cryptophane-A attached to a cysteine in extracellular loop 2 of the receptor facilitates chemical exchange saturation transfer experiments without and in the presence of native ligand neuropeptide Y. High-quality spectra indicative of structural states of the receptor-cage conjugate were obtained. Specifically, five signals could be assigned to the conjugate in the apo form. After the addition of NPY, one additional signal and subtle modifications in the persisting signals could be detected. The correlation of the spectroscopic signals and structural states was achieved with molecular dynamics simulations, suggesting frequent contact between the xenon trapping cage and the receptor surface but a preferred interaction with the bound ligand.

Rare Phytocannabinoids Exert Anti-Inflammatory Effects on Human Keratinocytes via the Endocannabinoid System and MAPK Signaling Pathway.

Increasing evidence supports the therapeutic potential of rare cannabis-derived phytocannabinoids (pCBs) in skin disorders such as atopic dermatitis, psoriasis, pruritus, and acne. However, the molecular mechanisms of the biological action of these pCBs remain poorly investigated. In this study, an experimental model of inflamed human keratinocytes (HaCaT cells) was set up by using lipopolysaccharide (LPS) in order to investigate the anti-inflammatory effects of the rare pCBs cannabigerol (CBG), cannabichromene (CBC), Δ9-tetrahydrocannabivarin (THCV) and cannabigerolic acid (CBGA). To this aim, pro-inflammatory interleukins (IL)-1β, IL-8, IL-12, IL-31, tumor necrosis factor (TNF-β) and anti-inflammatory IL-10 levels were measured through ELISA quantification. In addition, IL-12 and IL-31 levels were measured after treatment of HaCaT cells with THCV and CBGA in the presence of selected modulators of endocannabinoid (eCB) signaling. In the latter cells, the activation of 17 distinct proteins along the mitogen-activated protein kinase (MAPK) pathway was also investigated via Human Phosphorylation Array. Our results demonstrate that rare pCBs significantly blocked inflammation by reducing the release of all pro-inflammatory ILs tested, except for TNF-β. Moreover, the reduction of IL-31 expression by THCV and CBGA was significantly reverted by blocking the eCB-binding TRPV1 receptor and by inhibiting the eCB-hydrolase MAGL. Remarkably, THCV and CBGA modulated the expression of the phosphorylated forms (and hence of the activity) of the MAPK-related proteins GSK3β, MEK1, MKK6 and CREB also by engaging eCB hydrolases MAGL and FAAH. Taken together, the ability of rare pCBs to exert an anti-inflammatory effect in human keratinocytes through modifications of eCB and MAPK signaling opens new perspectives for the treatment of inflammation-related skin pathologies.

Insights into strigolactone (GR24) mediated regulation of cadmium-induced changes and ROS metabolism in Artemisia annua

Agricultural soil contamination and subsequently crops still require alternative solutions to reduce associated environmental risks. The effects of strigolactones (SLs) in alleviating cadmium (Cd) phytotoxicity in Artemisia annua plants were investigated during this study. Strigolactones play a vital role during plant growth and development due to their complex interplay during a plethora of biochemical processes. However, information on the potential of SLs to elicit abiotic stress signaling and trigger physiological modifications in plants is limited. In order to decipher the same, A. annua plants were exposed to different concentrations of Cd (20 and 40 mg kg-1), with or without the supplementation of exogenous SL (GR24, a SL analogue) at 4 µM concentration. Under Cd stress, excess Cd accumulation resulted in reduced growth, physio-biochemical traits, and artemisinin content. However, the follow-up treatment of GR24 maintained a steady state equilibrium between reactive oxygen species and antioxidant enzymes, improved chlorophyll fluorescence parameters such as Fv/Fm, ФPSII, and ETR for improved photosynthesis, enhanced chlorophyll content, maintained chloroplast ultrastructure, improved the glandular trichome (GT) attributes and artemisinin production in A. annua. Moreover, it also resulted in improved membrane stability, reduced Cd accumulation, and regulated the behaviour of stomatal apertures for better stomatal conductance under Cd stress. The results of our study suggest that GR24 could be highly effective in alleviating Cd-induced damages in A. annua. It acts via the modulation of the antioxidant enzyme system for redox homeostasis, protection of the chloroplasts and pigments for improved photosynthetic performance, and improved GT attributes for enhanced artemisinin production in A. annua.

Biosensors for landing creative intent

The motivation for use of biosensors in audiovisual media is made by highlighting problem of signal loss due to wide variability in playback devices. A metadata system that allows creatives to steer signal modifications as a function of audience emotion and cognition as determined by biosensor analysis.

Molecular Links between Flowering and Abiotic Stress Response: A Focus on Poaceae.

Extreme temperatures, drought, salinity and soil pollution are the most common types of abiotic stresses crops can encounter in fields; these variations represent a general warning to plant productivity and survival, being more harmful when in combination. Plant response to such conditions involves the activation of several molecular mechanisms, starting from perception to signaling, transcriptional reprogramming and protein modifications. This can influence the plant's life cycle and development to different extents. Flowering developmental transition is very sensitive to environmental stresses, being critical to reproduction and to agricultural profitability for crops. The Poacee family contains some of the most widespread domesticated plants, such as wheat, barley and rice, which are commonly referred to as cereals and represent a primary food source. In cultivated Poaceae, stress-induced modifications of flowering time and development cause important yield losses by directly affecting seed production. At the molecular level, this reflects important changes in gene expression and protein activity. Here, we present a comprehensive overview on the latest research investigating the molecular pathways linking flowering control to osmotic and temperature extreme conditions in agronomically relevant monocotyledons. This aims to provide hints for biotechnological strategies that can ensure agricultural stability in ever-changing climatic conditions.

Improved estimation of rail–wheel contact forces from instrumented wheel-set data through higher harmonic cancellation and a back-propagation neural network scheme

Instrumented wheelsets, equipped with numbers of strain gauges, are widely used to estimate the rail–wheel contact parameters. The accuracy in estimation of rail–wheel contact parameters from the measured strain signals depends on the layout of the strain gauges and correlation techniques adopted for processing measured signals. Harmonic components estimation is the most widely used conventional correlation technique. However, its accuracy in estimation of rail–wheel contact parameters is limited to the order of 80%–90%. This study proposes a signal modification algorithm, initially utilizing cancellation of next significant higher harmonics than those considered in the existing algorithms and then further, a novel Artificial Neural Network architecture for the same purpose.The signal modification approach is based on the realization that the seventh harmonic has significant contribution on the strain signals. The proposed algorithm is aimed at estimating and cancelling the effects of the seventh harmonic in strain signals. The accuracy thus improves to 91% in vertical contact force estimates, while the accuracy in estimation of lateral contact force and lateral contact position are improved to 97% and 95%, respectively. Due to the highly non-linear nature of the rail–wheel contact parameters, an optimized nonlinear mapping is required for further improvement in the accuracy.The viability of using artificial neural networks in estimation of rail–wheel contact parameters have been explored earlier. The proposed architectures are complicated and require extensive manual optimization of tuning parameters. A simple back-propagation artificial neural network architecture, using Bayesian regularization training function with Levenberg–Marquardt optimization technique is proposed in this study. The proposed neural network consists of 18 measured strain signals as input, three hidden layers with 20, 6 and 3 neurons, respectively, and vertical force, lateral force and lateral contact position as three outputs. Accuracies of the order of 99%, 99% and 96% are achieved in the estimation of vertical contact force, lateral contact force and lateral contact position respectively. The input and training data is generated from the multi-body dynamics and finite element simulations.