Final Effector Research Articles

Molecular mechanisms of angiotensin type 2 receptor-mediated nitric oxide pathway in angiotensin II-induced vasorelaxation: Roles of potassium channels.

A variety of biological functions is attributed to the renin-angiotensin system (RAS). One of them is regulating vascular tone through its final effector Angiotensin II (Ang II). Ang II action is mediated by the Angiotensin type 1 receptor (AT1-R) which plays a role in vasoconstriction, and Angiotensin type 2 receptor (AT2-R) which may result in vascular relaxation through the releasing of endothelium mediates relaxation factors such as Nitric Oxide (NO). Therefore, this study investigated the role of AT2-R in vasodilation after blocking the effect of AT1-R in the rat aorta. Furthermore, it is to determine whether or not Ang II through NO has a role in rat aorta dilation via using valsartan. For control isolated aortic rings were preincubated with Valsartan (AT1- R inhibitor) and then stimulated with angiotensin II dose-dependent. For treating aortic rings different blockers and inhibitors were used. Pd123177 (AT2- R inhibitor) (20 µM), an inhibitor of PKA H-89 (10 µM), eNOS inhibitor L-NAME (0.3 mM), with group of K channel blockers such as TEA (1 mM), 4-AP (1 mM), BaCl2 (1 mM), clotrimazole (0.03 mM) and GLIB (0.01 mM). Our analysis demonstrates vasodilation in aortic rings induced by Ang II after blocking ATI-R and this response was highly reliant on PKA/eNOS and cyclic guanosine monophosphate (cGMP). The data from this investigation provided evidence that Ca2 + activated K+ channels (KCa) and Voltage-dependent K channel (KV) mediated Ang II vasorelaxation. Finally, these results indicate that angiotensin II primarily induces dilatation AT2-R after inhibiting the angiotensin AT1 receptor through a cascade of signaling pathways involving many enzymes and plasma membrane protein channels.

SMURF1 and SMURF2 directly target GLI1 for ubiquitination and proteasome-dependent degradation

The transcription factor GLI1 is the main and final effector of the Hedgehog signaling pathway, which is involved in embryonic development, cell proliferation and stemness. Whether activated through canonical or non-canonical mechanisms, GLI1 aberrant activity is associated with Hedgehog-dependent cancers, including medulloblastoma, as well as other tumoral contexts. Notwithstanding a growing body of evidence, which have highlighted the potential role of post translational modifications of GLI1, the complex mechanisms modulating GLI1 stability and activity have not been fully elucidated. Here, we present a novel role played by SMURF1 and SMURF2 in the suppression of the Hedgehog/GLI signaling pathway through a direct targeting of GLI1. Indeed, the two SMURFs can interact with GLI1, exploiting the proline rich regions present on GLI1 protein, and trigger its polyubiquitination and proteasomal degradation, leading to a suppression of the Hedgehog pathway activity and a reduction of Hh-dependent tumor cell proliferation. Overall, this study adds new relevance to a tumor suppressive role of SMURFs on the Hedgehog pathway and confers upon them the status of potential therapeutic tools, either in canonical or non-canonical Hedgehog pathway aberrant activation.

A novel small molecule Enpp1 inhibitor improves tumor control following radiation therapy by targeting stromal Enpp1 expression

The uniqueness in each person’s cancer cells and variation in immune infiltrates means that each tumor represents a unique problem, but therapeutic targets can be found among their shared features. Radiation therapy alters the interaction between the cancer cells and the stroma through release of innate adjuvants. The extranuclear DNA that can result from radiation damage of cells can result in production of the second messenger cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) by cyclic GMP-AMP synthase (cGAS). In turn, cGAMP can activate the innate sensor stimulator of interferon genes (STING), resulting in innate immune activation. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1) is a phosphodiesterase that can be expressed by cancer cells that can degrade cGAMP, thus can decrease or block STING activation following radiation therapy, impairing the innate immunity that is critical to support adaptive immune control of tumors. We observed that many human and murine cancer cells lack Enpp1 expression, but that Enpp1 is expressed in cells of the tumor stroma where it limits tumor control by radiation therapy. We demonstrate in preclinical models the efficacy of a novel Enpp1 inhibitor and show that this inhibitor improves tumor control by radiation even where the cancer cells lack Enpp1. This mechanism requires STING and type I interferon (IFN) receptor expression by non-cancer cells and is dependent on CD8 T cells as a final effector mechanism of tumor control. This suggests that Enpp1 inhibition may be an effective partner for radiation therapy regardless of whether cancer cells express Enpp1. This broadens the potential patient base for whom Enpp1 inhibitors can be applied to improve innate immune responses following radiation therapy.

Stem cell factor-mediated upregulation of SIRT1 protects melanin-deprived keratinocytes against UV-induced DNA damage in individuals with vitiligo.

Despite the loss of melanocytes, individuals with vitiligo have a significantly lower risk of developing skin malignancies compared to ethnicity-matched controls. The study investigated the molecular mechanisms that protect skin cells (keratinocytes) from UV-B-induced DNA damage in individuals with vitiligo. The study found that upregulation of stem cell factor (SCF) signaling significantly reduced γ-H2AX positivity and cyclobutane pyrimidine dimer formation and improved mitochondrial health (elongated mitochondria, reduced reactive oxygen species [ROS] and lipid peroxidation) in keratinocytes upon UV-B exposure. Interestingly, SCF treatment also reduced lipid droplet accumulation and triacylglyceride levels by upregulating lipoprotein lipase (LPL). Further, siLPL increased DNA damage and lipid droplet (LD) accumulation, while NO-1886, an LPL agonist, reversed both, suggesting a direct link between lipid metabolism and DNA damage. Downregulation of NAD-dependent deacetylase sirtuin1 (SIRT1) with siRNA or with Ex-527, a pharmacological inhibitor of SIRT1, diminished the protective effects mediated by SCF and NO-1886, suggesting SIRT1 to be the final effector protein in the SCF-LPL-SIRT1 signaling axis. Analysis of clinical samples of vitiligo corroborated the upregulation of SCF and LPL in lesional epidermis. In conclusion, our study demonstrates a novel SCF-LPL-SIRT1 signaling axis that confers protection to vitiligo keratinocytes from the harmful effects of UV-B radiation.

Impact of ciprofloxacin with autophagy on renal tubular injury.

Renal tubular injury caused by oxidative stress and inflammation results in acute kidney injury. Recent research reported that antibiotics may protect renal tubules from progressive deterioration, but the underlying mechanism remains unclear. Therefore, we investigated the efficacy and mechanism of action of antibiotics against renal tubular injury. We screened ciprofloxacin, ceftizoxime, minocycline, and netilmicin and selected ciprofloxacin to examine further because of its low toxicity towards renal tubular cells. We evaluated the effect of ciprofloxacin on cell survival by analyzing apoptosis and autophagy. Terminal deoxynucleotidyl transferase-mediated d-UTP nick end labeling (TUNEL) assay results showed that the ciprofloxacin group had less apoptotic cells than the control group. The ratio of cleaved caspase 3 to caspase 3, the final effector in the apoptosis process, was decreased, but the ratio of B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) to Bcl-2 located upstream of caspase 3 was not decreased in the ciprofloxacin group. Therefore, apoptosis inhibition does not occur via Bax/Bcl-2. Conversely, the levels of phosphorylated Bcl-2, and Beclin-1, an autophagy marker, were increased, and that of caspase-3 was decreased in the ciprofloxacin group. This indicates that ciprofloxacin enhances autophagy, increasing the amount of free Beclin-1 via phosphorylated Bcl-2, and inhibits caspase activity. Therefore, ciprofloxacin might protect against renal tubular injury through the activation of autophagy in the setting of acute kidney injury.

Computational design and experimental confirmation of a disulfide-stapled YAP helixα1-trap derived from TEAD4 helical hairpin to selectively capture YAP α1-helix with potent antitumor activity.

Human Hippo signaling pathway is an evolutionarily conserved regulator network that controls organ development and has been implicated in various cancers. Transcriptional enhanced associate domain-4 (TEAD4) is the final nuclear effector of Hippo pathway, which is activated by Yes-associated protein (YAP) through binding to two separated YAP regions of α1-helix and Ω-loop. Previous efforts have all been addressed on deriving peptide inhibitors from the YAP to target TEAD4. Instead, we herein attempted to rationally design a so-called 'YAP helixα1-trap' based on the TEAD4 to target YAP by using dynamics simulation and energetics analysis as well as experimental assays at molecular and cellular levels. The trap represents a native double-stranded helical hairpin covering a specific YAP-binding site on TEAD4 surface, which is expected to form a three-helix bundle with the α1-helical region of YAP, thus competitively disrupting TEAD4-YAP interaction. The hairpin was further stapled by a disulfide bridge across its two helical arms. Circular dichroism characterized that the stapling can effectively constrain the trap into a native-like structured conformation in free state, thus largely minimizing the entropy penalty upon its binding to YAP. Affinity assays revealed that the stapling can considerably improve the trap binding potency to YAP α1-helix by up to 8.5-fold at molecular level, which also exhibited a good tumor-suppressing effect at cellular level if fused with TAT cell permeation sequence. In this respect, it is considered that the YAP helixα1-trap-mediated blockade of Hippo pathway may be a new and promising therapeutic strategy against cancers.

MDB-57. PROTEOMIC ANALYSIS REVEALS AMBRA1 AS A NEW CRL3REN/KCTD11 SUBSTRATE PROMOTING SONIC HEDGEHOG-DEPENDENT MEDULLOBLASTOMA

Abstract BACKGROUND Sonic Hedgehog (SHH) signaling pathway is fundamental for a proper embryonic development and its uncontrolled activation is responsible for various cancers. One the most relevant SHH-dependent tumors is medulloblastoma (MB), a highly heterogeneous brain malignancy that predominantly occurs in childhood. Understanding the complex networks that govern SHH aberrant activation represents a dramatic challenge for the development of efficient personalized therapies. Here, we identify a new role of the pro-autophagic protein AMBRA1 as interactor of REN/KCTD11 (here REN), the substrate-receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and tumor suppressor lost in ~30% of human SHH-MBs. We show a novel mechanism of action of AMBRA1 in SHH-MB pathogenesis whereby its highly expression, due to loss of REN, drives the activation of GLI1, the final effector of SHH signaling. METHODS Proteomic analysis, co-immunoprecipitation, ubiquitylation and cycloheximide assays have been performed to demonstrate that AMBRA1 is a substrate of ubiquitylation of CRL3REN and to define AMBRA1 mechanism of action in SHH-MB tumorigenesis. Transcriptomic analysis, in vitro and in vivo proliferation assays have been carried out on murine and patient-derived xenograft (PDX) models to examine the effect of AMBRA1 modulation on SHH-MB growth. RESULTS We demonstrate that CRL3REN binds and induces polyubiquitylation and proteasome-mediated degradation of AMBRA1. Interestingly, we found that genetic depletion of AMBRA1 strongly reduces SHH-dependent MB growth both in vitro and in vivo, an effect associated with the decrease in the expression of GLI1 and other SHH target genes. We hypothesize that AMBRA1 could impact on the stability of GLI1 by counteracting the activity of E3 ubiquitin ligases regulating GLI proteins. Remarkably, transcriptomic analysis of SHH PDX upon AMBRA1 knockdown revealed an impairment of SHH activity and an upregulation of cerebellum differentiation. CONCLUSION Our findings identify AMBRA1 as a novel CRL3REN substrate and promising therapeutic target for SHH-dependent cancer.

MDB-28. THE CYTOSKELETON REGULATORS FORMINS CONTROL THE SHH PATHWAY AND ARE INVOLVED IN MEDULLOBLASTOMA TUMORIGENESIS

Abstract BACKGROUND Medulloblastoma (MB) is a lethal pediatric malignancy of the cerebellum. Among MB’s molecular subgroups, Sonic Hedgehog (SHH) is the most abundant and it is characterized by alterations of key components of the SHH development pathway. However, the molecular mechanisms driving SHH-MB require to further be unveiled to design more effective therapies. Recently, we identified formins, well-known players of cytoskeletal dynamics, as regulators of SHH signaling able to affect SHH-MB growth. METHODS Luciferase functional assays, western-blot and RT-qPCR have been performed to analyze the effect of formins on GLI1 (the final effector of SHH signaling) transcriptional activity and expression. Proliferation assays and Brillouin microscopy have been carried out on primary murine SHH-MB cells to test the effect of formins on SHH-MB growth and stiffness. RESULTS We demonstrated that formins have a dual role in the regulation of SHH signaling, either positive or negative. The modulation of formins expression in SHH-dependent cell models affect GLI1 transcriptional activity and expression. Moreover, formins show opposite protein expression levels during the development of murine cerebellum, and their modulation impair the proliferation of granule cell progenitors (GCPs), the cells of origin of MB. Interestingly, formins protein levels are highly altered in SHH-MB samples. Notably, the overexpression or the genetic silencing of formins in SHH-MB primary cells from Math1-Cre/Ptc1fl/fl mice significantly impact on the cell proliferation as consequence of modulated GLI1 expression levels and the stiffness of primary SHH-MB cells, thus suggesting that formins could affect tumor cell motility and invasiveness. CONCLUSIONS Our findings identify formins as regulators of SHH signaling and illuminate on the role of cytoskeleton in SHH-MB for the design of innovative interventions.

Gasdermin D drives focal crystalline thrombotic microangiopathy by accelerating immunothrombosis and necroinflammation

AbstractThrombotic microangiopathy (TMA) is characterized by immunothrombosis and life-threatening organ failure but the precise underlying mechanism driving its pathogenesis remains elusive. In this study, we hypothesized that gasdermin D (GSDMD), a pore-forming protein that serves as the final downstream effector of the pyroptosis/interleukin-1β (IL-1β) pathway, contributes to TMA and its consequences by amplifying neutrophil maturation and subsequent necrosis. Using a murine model of focal crystalline TMA, we found that Gsdmd deficiency ameliorated immunothrombosis, acute tissue injury, and failure. Gsdmd−/− mice exhibited a decrease in mature IL-1β, as well as in neutrophil maturation, β2-integrin activation, and recruitment to TMA lesions, in which they formed reduced neutrophil extracellular traps in both arteries and interstitial tissue. The GSDMD inhibitor disulfiram dose-dependently suppressed human neutrophil pyroptosis in response to cholesterol crystals. Experiments with GSDMD–deficient, human–induced, pluripotent stem cell–derived neutrophils confirmed the involvement of GSDMD in neutrophil β2-integrin activation, maturation, and pyroptosis. Both prophylactic and therapeutic administration of disulfiram protected the mice from focal TMA, acute tissue injury, and failure. Our data identified GSDMD as a key mediator of focal crystalline TMA and its consequences, including ischemic tissue infarction and organ failure. GSDMD could potentially serve as a therapeutic target for the systemic forms of TMA.

Characterization of Human B Cell Hematological Malignancies Using Protein-Based Approaches.

The maturation of B cells is a complex, multi-step process. During B cell differentiation, errors can occur, leading to the emergence of aberrant versions of B cells that, finally, constitute a malignant tumor. These B cell malignancies are classified into three main groups: leukemias, myelomas, and lymphomas, the latter being the most heterogeneous type. Since their discovery, multiple biological studies have been performed to characterize these diseases, aiming to define their specific features and determine potential biomarkers for diagnosis, stratification, and prognosis. The rise of advanced -omics approaches has significantly contributed to this end. Notably, proteomics strategies appear as promising tools to comprehensively profile the final molecular effector of these cells. In this narrative review, we first introduce the main B cell malignancies together with the most relevant proteomics approaches. Then, we describe the core studies conducted in the field and their main findings and, finally, we evaluate the advantages and drawbacks of flow cytometry, mass cytometry, and mass spectrometry for the profiling of human B cell disorders.

Optogenetic induction of chronic glucocorticoid exposure in early-life leads to blunted stress-response in larval zebrafish.

Early life stress (ELS) exposure alters stress susceptibility in later life and affects vulnerability to stress-related disorders, but how ELS changes the long-lasting responsiveness of the stress system is not well understood. Zebrafish provides an opportunity to study conserved mechanisms underlying the development and function of the stress response that is regulated largely by the neuroendocrine hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis, with glucocorticoids (GC) as the final effector. In this study, we established a method to chronically elevate endogenous GC levels during early life in larval zebrafish. To this end, we employed an optogenetic actuator, beggiatoa photoactivated adenylyl cyclase, specifically expressed in the interrenal cells of zebrafish and demonstrate that its chronic activation leads to hypercortisolaemia and dampens the acute-stress evoked cortisol levels, across a variety of stressor modalities during early life. This blunting of stress-response was conserved in ontogeny at a later developmental stage. Furthermore, we observe a strong reduction of proopiomelanocortin (pomc)-expression in the pituitary as well as upregulation of fkbp5 gene expression. Going forward, we propose that this model can be leveraged to tease apart the mechanisms underlying developmental programming of the HPA/I axis by early-life GC exposure and its implications for vulnerability and resilience to stress in adulthood.

Dihydrotanshinone I exhibits antitumor effects via β-catenin downregulation in papillary thyroid cancer cell lines

Thyroid cancer is the most common endocrine carcinoma and, among its different subtypes, the papillary subtype (PTC) is the most frequent. Generally, PTCs are well differentiated, but a minor percentage of PTCs are characterized by a worse prognosis and more aggressive behavior. Phytochemicals, naturally found in plant products, represent a heterogeneous group of bioactive compounds that can interfere with cell proliferation and the regulation of the cell cycle, taking part in multiple signaling pathways that are often disrupted in tumor initiation, proliferation, and progression. In this work, we focused on 15,16-dihydrotanshinone I (DHT), a tanshinone isolated from Salvia miltiorrhiza Bunge (Danshen). We first evaluated DHT biological effect on PTC cells regarding cell viability, colony formation ability, and migration capacity. All of these parameters were downregulated by DHT treatment. We then investigated gene expression changes after DHT treatment by performing RNA-seq. The analysis revealed that DHT significantly reduced the Wnt signaling pathway, which plays a role in various diseases, including cancer. Finally, we demonstrate that DHT treatment decreases protein levels of β-catenin, a final effector of canonical Wnt signaling pathway. Overall, our data suggest a possible use of this nutraceutical as an adjuvant in the treatment of aggressive papillary thyroid carcinoma.

Design, synthesis and anti-necroptosis activity of fused heterocyclic MLKL inhibitors

Necroptosis is an important form of programmed cell death (PCD), which is mediated by a death receptor and independent of the caspase proteolytic enzyme. Mixed lineage kinase domain-like (MLKL) is the final effector of necroptosis, playing an irreplaceable role in the execution of necroptosis. However, the studies on MLKL inhibitors are in their infancy. Necrosulfonamide (NSA) is an early-discovered covalent MLKL inhibitor, possessing medium anti-necroptosis activity and a structure-activity relationship (SAR) not widely disclosed. In this study, with the covalent motif maintained, we aim to improve the activity by introducing the terminal fused heterocycles and meanwhile revealing the SAR on the part. As a result, compounds 9 and 14 showed the best activity (EC50 = 148.4 and 595.9 nM) against necroptosis among the analogues by covalently binding to MLKL. The SAR was also concluded to guide further structural optimization in this field.

Treatment development targeting Hippo pathway for scirrhous type gastric cancer.

398 Background: Treatment outcome of gastric cancer (GC) has been improved by minimally invasive surgery, and development of new drugs including immune checkpoint inhibitors. However, survival for patients with scirrhous type (SGC, Type4) gastric cancer is dismal. Methods: We collected miRNAs form exosomes released from cancer associated fibroblasts which is developed from resected gastric cancer tissue. Pathway enrichment analysis (PEA) of top miRNAs showed that they are significantly associated with Hippo signaling pathway (Hippo pathway). The molecules relating Hippo pathway was evaluated by Western blotting in cell line, and IHC of tissue array. Results: PEA using top miRNAs showed the significant relationship to Hippo pathway. Yap is a final effector in this pathway, and we found that YAP is not expressed in MKN45 cells (diffuse type), which is supposed to be degraded in cytoplasm. On the contrary, MKN74 cells express both YAP and pYAP. IHC showed that YAP translocation to nucleus is significantly associated with CTGF expression. CTGF low expression is significantly associated with worse prognosis especially in diffuse type GC (Log rank p = 0.022). This trend is similar in the analysis of patients with SGC. Conclusions: The role of Hippo pathway is different between intestinal type and diffuse type of GC. YAP activator could be effective for the treatment of SGC, and we are focusing on the treatment development.

Inverse Kinematics Proposal to Automatize the 3D Scanning of Handball Shoes by Using a Robotic Arm with 3 Actuated Joints

The purpose of this paper is to present a procedure for automating the scanning process based on a mathematical model for a handheld 3D scanner for shoes used in some indoor sports. The study aims to use inverse kinematics to automate 3d footwear scanning used for indoor sport (handball) using a robotic arm with three joints. A modeling of the robotic arm and final effector was performed, to simulate the minimum and maximum trajectory of the robot arm according to the angles shown based on the mathematical model and inverse kinematics. With an easy-to-use interface and ergonomic design, this 3D scanning solution offers the versatility to scan various objects (such as scanning two shoe models used in indoor sports) and complex surface types. This scanning manner represents the state of the art of 3D scanning solutions as well as a benchmark in the 3D measurement equipment industry. The data obtained as a result of this research provide new directions and solutions for sports shoe scanning for indoor sports based on scanning trajectories preset by inverse kinematics in order to automate the scanning process using a handheld 3D scanner. Based on the mathematical model presented in the paper, automation of the scanning process can be achieved by maintaining the proposed trajectory using an automated arm operating through a control program that can be run on a simple controller.

RANBP1, a member of the nuclear-cytoplasmic trafficking-regulator complex, is the terminal-striking point of the SGK1-dependent Th17+ pathological differentiation.

The Th17+ arrangement is critical for orchestrating both innate and acquired immune responses. In this context, the serum and glucocorticoid regulated kinase 1 (SGK1) exerts a key role in the governance of IL-23R-dependent Th17+ maturation, through the phosphorylation-dependent control of FOXO1 localization. Our previous work has shown that some of the SGK1-key functions are dependent on RAN-binding protein 1 (RANBP1), a terminal gene in the nuclear transport regulation. Here, we show that RANBP1, similarly to SGK1, is modulated during Th17+ differentiation and that RANBP1 fluctuations mediate the SGK1-dependent effects on Th17+ maturation. RANBP1, as the final effector of the SGK1 pathway, affects FOXO1 transport from the nucleus to the cytoplasm, thus enabling RORγt activation. In this light, RANBP1 represents the missing piece, in an essential and rate-limiting manner, underlying the Th17+ immune asset.

Aldosterone: Essential for Life but Damaging to the Vascular Endothelium.

The renin angiotensin aldosterone system is a key regulator of blood pressure. Aldosterone is the final effector of this pathway, acting predominantly via mineralocorticoid receptors. Aldosterone facilitates the conservation of sodium and, with it, water and acts as a powerful stimulus for potassium excretion. However, evidence for the pathological impact of excess mineralocorticoid receptor stimulation is increasing. Here, we discussed how in the heart, hyperaldosteronism is associated with fibrosis, cardiac dysfunction, and maladaptive hypertrophy. In the kidney, aldosterone was shown to cause proteinuria and fibrosis and may contribute to the progression of kidney disease. More recently, studies suggested that aldosterone excess damaged endothelial cells. Here, we reviewed how damage to the endothelial glycocalyx may contribute to this process. The endothelial glycocalyx is a heterogenous, negatively charged layer on the luminal surface of cells. Aldosterone exposure alters this layer. The resulting structural changes reduced endothelial reactivity in response to protective shear stress, altered permeability, and increased immune cell trafficking. Finally, we reviewed current therapeutic strategies for limiting endothelial damage and suggested that preventing glycocalyx remodelling in response to aldosterone exposure may provide a novel strategy, free from the serious adverse effect of hyperkalaemia seen in response to mineralocorticoid blockade.

Abstract 4869: Furin and Notch signaling pathway as part of the 2OHOA landscape against glioblastoma

Abstract Glioblastoma (GBM) is the most common and aggressive cancer tumor of the central nervous system, with only 12-15 months of patients’ median survival. Melitherapy is a novel therapeutic platform based on the regulation of the membrane’s structure and organization with the consequent modulation of certain cell signals. In this context, 2-hydroxioleic acid (2OHOA, LAM561, INN: idroxioleic acid) has been developed for the treatment of GBM and is currently running a phase IIB/III clinical trial for newly diagnosed GBM patients. 2OHOA modulates the lipid composition and structure of cancer cells, increasing membranes fluidity and altering the activity of membrane-associated proteins, inhibiting the proliferation, inducing ER stress and differentiation, and finally triggering cell death by autophagy. The Notch signaling pathway has been highly related to tumorigenesis and cell survival driving the pathogenesis of GBM. In this work, we studied whether 2OHOA modulates the Notch pathway and its relevance in its mechanism of action (MoA) as an antitumoral drug. For this purpose, the 2OHOA’s effect on different components of the pathway was studied by western-blot, Q-PCR, and confocal microscopy. In addition, Notch receptor processing and NICD formation (final effector) were analyzed by cell fractionation and Notch processing enzymes activity, like furin, was evaluated upon 2OHOA treatment. First, the inhibition of this pathway in GBM cells by 2OHOA was confirmed by (i) lower expression levels of some of its components (Notch1, Notch3, and Jagged) and target genes (Hes1, CD3), (ii) less nuclear presence of NICD and Hes1 after 2OHOA treatment, (iii) accumulation of full-length Notch receptor in the membranes, impairing its processing and the NICD formation. Second, the relevance of Notch pathway in 2OHOA’s MoA was determined by the partial hindering of 2OHOA antiproliferative effect by Hes1 overexpression. Moreover, the reduction of Hes1 expression induced by the drug in different GBM cells correlated positively with their sensitivity to 2OHOA. Finally, 2OHOA downregulated furin-like proteases activity by physical association, an enzyme responsible for the Notch processing first step. All together reveals that the inhibition of Notch pathway by 2OHOA plays a role in its antitumoral effect, and this event is unleashed by the direct furin enzyme inhibition, identifying it as a novel target for this drug in the GBM and other pathologies treatment. Citation Format: Raquel Rodríguez-Lorca, Roberto Beteta-Göbel, Ramón Román, Manuel Torres, Victoria Llado, Pablo V. Escribá, Paula Fernández-García. Furin and Notch signaling pathway as part of the 2OHOA landscape against glioblastoma. [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 4869.

Innate Immune Response of TmToll-3 Following Systemic Microbial Infection in Tenebrio molitor.

Although Toll-like receptors have been widely identified and functionally characterized in mammalian models and Drosophila, the immunological function of these receptors in other insects remains unclear. Here, we explored the relevant innate immune response of Tenebrio molitor (T. molitor) Toll-3 against Gram-negative bacteria, Gram-positive bacteria, and fungal infections. Our findings indicated that TmToll-3 expression was mainly induced by Candida albicans infections in the fat bodies, gut, Malpighian tubules, and hemolymph of young T. molitor larvae. Surprisingly, Escherichia coli systemic infection caused mortality after TmToll-3 knockdown via RNA interference (RNAi) injection, which was not observed in the control group. Further analyses indicated that in the absence of TmToll-3, the final effector of the Toll signaling pathway, antimicrobial peptide (AMP) genes and relevant transcription factors were significantly downregulated after E. coli challenge. Our results indicated that the expression of almost all AMP genes was suppressed in silenced individuals, whereas the expression of relevant genes was positively regulated after fungal injection. Therefore, this study revealed the immunological involvement of TmToll-3 in T. molitor in response to systematic infections.

Dynamic Analysis of Flexible two Link Robotic Arm Considering Joint Stiffness

The robotic manipulator is a machine that can perform a variety of tasks according to specifications without the need of human interference. In order to model and control such devices, vibration analysis of flexible manipulators has become a critical field of study. The finite element approach has been used to analyze single and double connection flexible manipulators made of advanced composite material in the current study. For the modelling and study of the versatile composite manipulators, a three-noded beam feature is used. The effects of the taper angles of tapered flexible composite manipulators on the final product effector movement and vibration has been considered. In present work CATIA V5 is used to model the flexible single link robotic arm and static structural analysis to find stress and total deformation, dynamic analysis is carried out to find different modes, corresponding frequency, and life estimation of flexible single and double link robotic arm and also material optimization is done using different material composition for structural steel, aluminium and CFRP composite material using ANSYS WORKBENCH 18.2 (Finite Element Approach) for life estimation and evaluation using analytical approach.