Significant Blockade Research Articles

Exceptional Field Effect and Negative Differential Conductance in Spiro-Conjugated Single-Molecule Junctions.

The advancement of molecular electronics endeavors to build miniaturized electronic devices using molecules as the key building blocks by harnessing their internal structures and electronic orbitals. To date, linear planar conjugated or cross-conjugated molecules have been extensively employed in the fabrication of single-molecule devices, benefiting from their good conductivity and compatibility with electrode architectures. However, the development of multifunctional single-molecule devices, particularly those with unique charge transport properties, necessitates a more rigorous selection of molecular materials. Among different assortments of molecules suited for the construction of molecular circuits, Spiro-conjugated structures, specifically spirobifluorene derivatives, stand out as promising candidates due to their distinctive electronic properties. In this work, we focus on the charge transport characteristics of Spiro-conjugated molecules sandwiched between graphene nanogaps. Experiments reveal significant Coulomb blockade and distinct negative differential conductance effects. Beyond two-terminal device measurements, solid-state gate electrodes are utilized to create single-molecule transistors, successfully modulating the molecular energy levels to achieve an on/off ratio exceeding 1000. This endeavor not only offers valuable insights into the design and fabrication of future practical molecular devices, blessed with enhanced performance and functionality, but also presents a new paradigm for the investigation of fundamental physical phenomena.

Angstrom-Level Tuning in Confined Space of Metal-Organic Framework for Ultra-High Resolution in Gas Chromatographic Separation.

Optimizing the balance between thermodynamic interaction and kinetic diffusion is pivotal to obtaining high-performance gas chromatographic stationary phases. Here, three aluminum-based metal-organic frameworks featuring fym topology were chosen to achieve such balance by refined controlling the thermodynamic interactions toward analytes at angstrom level in a confined space. The CAU-10-H with the middle-sized channels (5.4 Å) provided weak interactions with xylenes because of the benzene ring around the channel, leading to the fastest diffusion. While the MIL-160 provided stronger interactions toward the analytes due to the abundance of O-heterocyclic sites of 2,5-furandicarboxylic acid ligands, resulting in slightly higher diffusion barriers. Thereby, although MIL-160 had a larger channel (5.9 Å) than CAU-10-H, the xylenes still diffused more slowly in MIL-160 than in CAU-10-H. The CAU-10-NH2 with the channel of 4.7 Å provided overstrong thermodynamic interactions and significant stereospecific blockade to the analytes because of the NH2 sites in the confined channels. These factors collectively contributed to achieving the lowest diffusion kinetics. The confined interactions were also proved by molecular dynamics simulation. Furthermore, the application indicated that MIL-160 exhibited the highest separation ability as a GC stationary phase among all reported materials. This strategy offers an approach for developing high-performance MOF stationary phases.

Eomesodermin spatiotemporally orchestrates the early and late stages of NK cell development by targeting KLF2 and T-bet, respectively.

Eomesodermin (Eomes) is a critical factor in the development of natural killer (NK) cells, but its precise role in temporal and spatial coordination during this process remains unclear. Our study revealed that Eomes plays distinct roles during the early and late stages of NK cell development. Specifically, the early deletion of Eomes via the CD122-Cre transgene resulted in significant blockade at the progenitor stage due to the downregulation of KLF2, another important transcription factor. ChIP-seq revealed direct binding of Eomes to the conserved noncoding sequence (CNS) of Klf2. Utilizing the CHimeric IMmune Editing (CHIME) technique, we found that deletion of the CNS region of Klf2 via CRISPRi led to a reduction in the NK cell population and developmental arrest. Moreover, constitutive activation of this specific CNS region through CRISPRa significantly reversed the severe defects in NK cell development caused by Eomes deficiency. Conversely, Ncr1-Cre-mediated terminal deletion of Eomes expedited the transition of NK cell subsets from the CD27+CD11b+ phenotype to the CD27-CD11b+ phenotype. Late-stage deficiency of Eomes led to a significant increase in T-bet expression, which subsequently increased the expression of the transcription factor Zeb2. Genetic deletion of one allele of Tbx21, encoding T-bet, effectively reversed the aberrant differentiation of Eomes-deficient NK cells. In summary, we utilized two innovative genetic models to elucidate the intricate mechanisms underlying Eomes-mediated NK cell commitment and differentiation.

An Overview on Causes of Nonadherence in the Treatment of Rheumatoid Arthritis: Its Effect on Mortality and Ways to Improve Adherence.

Rheumatoid arthritis is one of the most prevalent musculoskeletal disorders that, when insufficiently treated, results in detrimental sequelae including joint damage and reduced quality of life. Poor patient adherence to medication is a significant blockade to effective management. The purpose of this review is to highlight and discuss the factors responsible for defiance of antirheumatic medication and ways to overcome these barriers. Education level, health literacy, cohabitation status, multi-morbidities, complicated drug regimen, intermittent co-payments, prescribed regimen adverse effects, and cognitive impairment are a few among many common barrier factors leading to poorer outcomes in rheumatoid arthritis. While there is an abundance of inhibitory factors leading to worsening disease progression, they each can be easily dealt with an effective approach at the beginning or during the treatment course to ensure a better outcome.

Comparación del bloqueo del grupo de nervios pericapsulares (PENG) y la analgesia epidural después de una artroplastia total de cadera: análisis retrospectivo

Introduction and objectivesTotal hip arthroplasty (THA) is an increasingly common orthopedic pro-cedure, with moderate to severe postoperative pain. Pericapsular nerve group (PENG) block is a recent block that seems to provide adequate analgesia without significant motor blockade. The aim of this study is to retrospectively compare the analgesic efficacy and safety of PENG block with those of epidural analgesia, in patients undergoing THA. Material and methodsThis is a retrospective observational study of patients who underwent primary THA, submitted to epidural analgesia or single-shot ultrasound-guided PENG block, during a one-year period. Data regarding demographic characteristics, surgery and anesthesia techniques, pain scores, opioid consumption, complications and time to hospital discharge were retrieved from institutional records and compared between the 2 groups (epidural analgesia vs PENG block). ResultsNo significant difference was found regarding pain scores, opioid consumption, and mean time to hospital discharge between the 2 groups. Pain scores at rest (1.20 epidural vs 1.67 PENG) or with movement (3.95 epidural vs 3.72 PENG) were similar between groups. Total number of complications was higher in the epidural analgesia group (50% epidural vs 5% PENG). Paresthesia was reported in both groups. Motor block, sedation, nausea and catheter-related complications were only found in the epidural analgesia group. ConclusionsPENG block seems to be equivalent to epidural analgesia regarding quality of postop-erative analgesia for patients subject to primary THA, supporting routine use of this block in these patients. The low rate of reported complications limits conclusions on this topic.

Ropivacaine-loaded, hydroxypropyl chitin thermo-sensitive hydrogel combined with hyaluronan: an injectable, sustained-release system for providing long-lasting local anesthesia in rats

Background and objectiveRopivacaine hydrochloride is a commonly used local anesthetic in clinics. However, local injection or continuous infusion of ropivacaine has been associated with several disadvantages. Accordingly, it is important...

Inhibition of protein N-myristoylation blocks Plasmodium falciparum intraerythrocytic development, egress and invasion.

We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of “pseudoschizonts,” which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.

Development of a triazolobenzodiazepine-based PET probe for subtype-selective vasopressin 1A receptor imaging

ObjectivesTo enable non-invasive real-time quantification of vasopressin 1A (V1A) receptors in peripheral organs, we sought to develop a suitable PET probe that would allow specific and selective V1A receptor imaging in vitro and in vivo. MethodsWe synthesized a high-affinity and -selectivity ligand, designated compound 17. The target structure was labeled with carbon-11 and tested for its utility as a V1A-targeted PET tracer by cell uptake studies, autoradiography, in vivo PET imaging and ex vivo biodistribution experiments. ResultsCompound 17 (PF-184563) and the respective precursor for radiolabeling were synthesized in an overall yield of 49% (over 7 steps) and 40% (over 8 steps), respectively. An inhibitory constant of 0.9 nM towards the V1A receptors was measured, while excellent selectivity over the related V1B, V2 and OT receptor (IC50 >10,000 nM) were obtained. Cell uptake studies revealed considerable V1A binding, which was significantly reduced in the presence of V1A antagonists. Conversely, there was no significant blockade in the presence of V1B and V2 antagonists. In vitro autoradiography and PET imaging studies in rodents indicated specific tracer binding mainly in the liver. Further, the pancreas, spleen and the heart exhibited specific binding of [11C]17 ([11C]PF-184563) by ex vivo biodistribution experiments. ConclusionWe have developed the first V1A-targeted PET ligand that is suitable for subtype-selective receptor imaging in peripheral organs including the liver, heart, pancreas and spleen. Our findings suggest that [11C]PF-184563 can be a valuable tool to study the role of V1A receptors in liver diseases, as well as in cardiovascular pathologies.

High sensitivity of deoxyribonucleic acid detection via graphene nanohole/silicon micro-nanopore structure fabricated by focused ion beam

Detection via nanohole is a simple sensing tool to analyze various biopolymers with high sensitivity. This paper reports the detection of deoxyribonucleic acid (DNA) biomolecules using an integration of 30 nm circular-shaped graphene nanohole and conical-shaped silicon (Si) micro-nanopore fabricated by focused ion beam technique. The ionic current changes at different DNA volume measured via a single and an array of nanoholes seem to confirm the ionic current blockade phenomena where the current decreases in stable linearity with the increase of volume. Regardless of the number of nanoholes, the sensitivity of around 110 nA/µL for both devices shows relatively much higher value as compared to the sensitivity reported in the other literatures. Such significant ionic current blockade detection suggests that a proposed combination of conical-shaped Si micro-nanopore and graphene nanohole are able to act as a promising fluidic channel and sensing membrane, respectively, towards a realization of high sensitive DNA biosensors.

Ferredoxin reductase regulates proliferation, differentiation, cell cycle and lipogenesis but not apoptosis in SZ95 sebocytes

Ferredoxin reductase (FDXR), a mitochondrial membrane-associated flavoprotein, is essential for electron transfer and modulates p53-dependent apoptosis in cancer cells.FDXR may be implicated in epidermal and sebocytic differentiation, but its explicit function in sebocytes remains to be elucidated. In the present study, immunohistochemistry revealed that FDXR expression was increased in sebaceous cells of acne lesions. FDXR, PPARγ, LXRα/β, SREBP1 and Sox9 expression was incremental during sebocyte differentiation. FDXR overexpression induced by Ad-GFP-FDXR infection enhanced differentiation, reactive oxygen species (ROS), lipogenesis and PPARγ expression, and consequnently inhibited proliferation in SZ95 sebocytes. Flow cytometry showed that FDXR overexpression induced significant blockade of G2/M phase but had no effect on sub-G1 (apoptotic) sebocytes. Insulin-like growth factor-1 (IGF-1)-induced FDXR and PPARγ expression and lipogenesis were abolished by pretreatment with PI3K inhibitor LY294002. These results suggest that FDXR overexpression might promote differentiation and lipogenesis via ROS production and suppress proliferation via G2/S blockade in SZ95 sebocytes. IGF-1 could facilitate differentiation and lipogenesis through PI3K/Akt/FDXR pathway. FDXR could serve as a potential marker of advanced sebaceous differentiation, and its overexpression may be involved in the development of acne lesions.

INFLAMMATORY CYTOKINES AND MICROBIAL LIGANDS AND METABOLITES INTERACT TO MODULATE DUOX2 EXPRESSION AND ACTIVITY

Abstract Background The pathogenesis of inflammatory bowel diseases (IBD) is characterized by a dysregulated crosstalk between the host and the microbiome that leads to the development of inflammation and dysbiosis. Dysbiosis in IBD involves an expansion of Proteobacteria and a reduction of Firmicutes, particularly of butyrate-producing species such as Faecalibacterium prausnitzii. The epithelial NADPH oxidase dual oxidase 2 (DUOX2), which prevents bacterial colonization of the mucosa through the production of hydrogen peroxide (H2O2), is the only gene consistently altered in IBD patients before the onset of disease. However, the involvement of DUOX2 in IBD is not well understood. We aimed to define how inflammation and the microbiota regulate DUOX2 activity. Methods C57BI/6J males raised in specific-pathogen free (SPF) and germ-free (GF) conditions underwent a model of dextran sulfate sodium (DSS)-induced colitis for 6 days. We obtained colon specimens for histopathology and isolation of colon epithelial cells (CEC). We stimulated colonoids from wild-type (WT), toll-like receptor 4-KO (Tlr4-KO), and Duox2-KO mice with IFNγ, heat-killed adherent invasive Escherichia coli (AIEC) strain LF82, and heat-killed F. prausnitzii strain A2-165 and determined expression of Duox2, Duoxa2, and production of H2O2. We also treated colonoids with butyrate, a microbial metabolite with anti-inflammatory properties. Extracellular H2O2 production was analyzed by means of the Amplex Red assay, whereas gene expression was determined by qPCR. Results SPF mice undergoing DSS-induced colitis developed overt inflammation that was accompanied by upregulation of Duox2 and Duoxa2, as well as increased production of H2O2 in freshly isolated CECs. DSS-treated GF mice developed a mild inflammation that also caused increased H2O2 production and Duoxa2 upregulation. WT colonoid stimulation with IFNγ and the Crohn’s disease-associated pathobiont AIEC induced Duox2 and Duoxa2 expression, whereas heat-killed F. prausnitzii did not. Similarly, both IFNγ and AIEC promoted epithelial production of H2O2 in WT colonoids but not Duox2-KO colonoids, indicating that epithelial release of H2O2 in response to these stimuli is mediated by DUOX2. Response to AIEC additionally required functional TLR4. Although heat-killed F. prausnitzii did not alter H2O2 production, its metabolite butyrate caused a significant blockade in the release of H2O2 in response to both IFNγ and AIEC. Conclusions Our results show that both inflammation and pathobionts induce the expression and activity of DUOX2, which begets more inflammation. We posit that specific depletion of pathobionts or restitution with butyrate-producing bacteria such as F. prausnitzii may be beneficial in IBD.

Proteins from SARS-CoV-2 reduce T cell proliferation: A mirror image of sepsis

Increased cytokine levels, acute phase reactants and immune checkpoint expression changes have been described in patients with Coronavirus Disease 2019 (COVID-19). Here, we have reported a monocyte polarization towards a low HLA-DR and high PD-L1 expression after long exposure to proteins from SARS-CoV-2. Moreover, CD86 expression was also reduced over SARS-CoV-2 proteins exposure. Additionally, T-cells proliferation was significantly reduced after stimulation with these proteins. Eventually, patients with long-term SARS-CoV-2 infection also exhibited a significant blockade of T-cells proliferation.

A Prospective Double-Blind Comparative Clinical Study Between Caudal Levobupivacaine (0.125%) with Clonidine and Ropivacaine (0.125%) with Clonidine on Post-Operative Analgesia in Paediatric Patients Undergoing Infra-Umbilical Surgery.

IntroductionCaudal epidural block is a reliable technique in paediatric patients but associated with various complications especially with higher concentration of drugs. We proposed a comparative study between levobupivacaine and ropivacaine at low concentration (0.125%) with clonidine at low dose (1 mcg/kg) taken as adjuvant. We aimed to see duration of post-operative analgesia, degree of motor blockade and other associated complications.Materials and MethodsEighty paediatric patients (1–6 years), American society of anaesthesiologists grade I and II, undergoing infra-umbilical surgery under general anaesthesia were randomly allocated into two groups of 40 each. Group A patients were given caudal levobupivacaine (0.125%) and Group B patients were given caudal ropivacaine (0.125%). Clonidine (1 mcg/kg) was taken as adjuvant in both the groups. Post-operative pain, sedation and motor blockade were assessed at 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 18 hours and 24 hours using Observational Pain Scale, modified Bromage Scale and four-point sedation score, respectively. Any other complications were also noted.ResultsMotor blockade was not associated with any of the patients. Duration of post-operative sedation was similar in both the groups. Duration of post-operative analgesia was significantly higher in Group A (p < 0.0001). Adverse effects and complications were negligible in both the groups.ConclusionBoth levobupivacaine and ropivacaine can be used safely at low concentration (0.125%) taking clonidine at low dose (1 mcg/kg) as adjuvant in paediatric caudal epidural block without significant motor blockade and other complications, duration of post-operative analgesia being significantly higher in the levobupivacaine group.

A comparison of fetomaternal outcome in PCEA using fentanyl, clonidine and dexmedetomidine as adjuvants with Ropivacaine in painless labor: a prospective, double blinded randomized study

Background: Patient controlled epidural analgesia has been associated with marked maternal satisfaction. Combination of local anesthetics with various adjuvants have been tried to ensure optimum analgesia with no or negligible fetomaternal side effects. Aim: To compare fentanyl, clonidine or dexmedetomidine as adjuvants with ropivacaine for labor epidural analgesia (LEA) using a PCEA pump with the objective to assess fetomaternal outcome in terms of analgesic effect, success rate of vaginal delivery, complications, neonatal APGAR score and maternal satisfaction. Materials and methods: Sixty full term laboring women received 10 ml 0.2% ropivacaine followed by continuous infusion of 0.1% ropivacaine with 2 μg/ml of either dexmedetomidine, fentanyl or clonidine respectively in Groups A, B, and C at 6 ml/hr. demand bolus setting was 2 ml with a lock out interval of 15 minutes. At full cervical dilatation another 10 ml bolus of respective solution were given. Parturients were monitored at 0, 10, 20, 30 min after giving 1st epidural bolus dose and then at 30 min interval for ongoing labor for pain relief (VAS), motor blockade (Bromage score), progress of labor (duration of 1st stage and 2nd stage), mode of delivery, fetal APGAR score (at 1 min and 5 min), vitals (HR, NIBP, RR, SpO2), overall patient satisfaction and complications. The statistical analysis was done both qualitatively (Fisher-exact test/Chi-square test) and quantitatively (one-way analysis of variance test with post-hoc intergroup comparisons using Bonferroni’s correction). Results: Onset of pain relief was earlier in fentanyl group, however after 1 h all three groups showed comparable pain relief (P&gt;0.05). There was a significant reduction in HR in group C and B compared to group A (P&lt;0.001) and MAP in group C compared to groups A and B. The motor- blocking potency was slightly higher in dexmedetomidine group, however no significant motor weakness observed in any parturient. Mean demand bolus need was more in group C compared to A and B (P&lt;0.001). There was no significant difference in mode of delivery (either SVD or cesarean) in between the groups. There was not a single case of fetal distress and most of the parturients showed satisfactory response to PCEA. Conclusion: All three study drugs produced equipotent analgesia in combination with ropivacaine 0.1%. There was absolute pain relief without significant motor blockade or any increase in instrumentation/cesarean deliveries or any adverse fetal outcomes.

Evidence-based Review of Periarticular Injections and Peripheral Nerve Blocks in Total Knee Arthroplasty

Background: Recently, post-operative pain management after Total Knee Arthroplasty (TKA) has focused on a multimodal approach for reducing opioid requirements, promoting early rehabilitation, and expediting discharge from hospital. Regional anesthesia, in the form of Periarticular Injections (PAI) and Peripheral Nerve Blocks (PNB), has shown promise as adjunctive therapy to oral analgesics. Objective: To review the current literature surrounding regional anesthesia for TKA. Discussion: PNBs provide effective analgesia after TKA. Historically, femoral nerve blocks (FNB) have been commonly employed. FNBs, however, lead to the significant motor blockade to the quadriceps musculature, which can dampen early rehabilitation efforts and increase the risk of post-operative falls. Adductor Canal Blocks (ACB) have shown excellent results in reducing post-operative pain while minimizing motor blockade. Periarticular injections (PAI), and infiltration between the popliteal Artery and Capsule of the Knee (IPACK) have similarly helped in reducing patient discomfort after TKA and providing analgesia to the posterior capsular region of the knee. Conclusion: PAIs, and PNBs are important elements in many multimodal postoperative pain management protocols after TKA. Current evidence appears to suggest that a combination of an ACB supplemented by posterior capsular analgesic coverage through PAI or IPACK may provide optimal results.

The voltage-gated sodium channel inhibitor, 4,9-anhydrotetrodotoxin, blocks human Nav1.1 in addition to Nav1.6

Voltage-gated sodium channels (VGSCs) are responsible for the initiation and propagation of action potentials in neurons. The human genome includes ten human VGSC α-subunit genes, SCN(X)A, encoding Nav1.1–1.9 plus Nax. To understand the unique role that each VGSC plays in normal and pathophysiological function in neural networks, compounds with high affinity and selectivity for specific VGSC subtypes are required. Toward that goal, a structural analog of the VGSC pore blocker tetrodotoxin, 4,9-anhydrotetrodotoxin (4,9-ah-TTX), has been reported to be more selective in blocking Na+ current mediated by Nav1.6 than other TTX-sensitive VGSCs, including Nav1.2, Nav1.3, Nav1.4, and Nav1.7. While SCN1A, encoding Nav1.1, has been implicated in several neurological diseases, the effects of 4,9-ah-TTX on Nav1.1-mediated Na+ current have not been tested. Here, we compared the binding of 4,9-ah-TTX for human and mouse brain preparations, and the effects of 4,9-ah-TTX on human Nav1.1-, Nav1.3- and Nav1.6-mediated Na+ currents using the whole-cell patch clamp technique in heterologous cells. We show that, while 4,9-ah-TTX administration results in significant blockade of Nav1.6-mediated Na+ current in the nanomolar range, it also has significant effects on Nav1.1-mediated Na+ current. Thus, 4,9-ah-TTX is not a useful tool in identifying Nav1.6-specific effects in human brain networks.

Evaluation of 18F-IAM6067 as a sigma-1 receptor PET tracer for neurodegeneration in vivo in rodents and in human tissue.

The sigma 1 receptor (S1R) is widely expressed in the CNS and is mainly located on the endoplasmic reticulum. The S1R is involved in the regulation of many neurotransmission systems and, indirectly, in neurodegenerative diseases. The S1R may therefore represent an interesting neuronal biomarker in neurodegenerative diseases such as Parkinson's (PD) or Alzheimer's diseases (AD). Here we present the characterisation of the S1R-specific 18F-labelled tracer 18F-IAM6067 in two animal models and in human brain tissue.Methods: Wistar rats were used for PET-CT imaging (60 min dynamic acquisition) and metabolite analysis (1, 2, 5, 10, 20, 60 min post-injection). To verify in vivo selectivity, haloperidol, BD1047 (S1R ligand), CM398 (S2R ligand) and SB206553 (5HT2B/C antagonist) were administrated for pre-saturation studies. Excitotoxic lesions induced by intra-striatal injection of AMPA were also imaged by 18F-IAM6067 PET-CT to test the sensitivity of the methods in a well-established model of neuronal loss. Tracer brain uptake was also verified by autoradiography in rats and in a mouse model of PD (intrastriatal 6-hydroxydopamine (6-OHDA) unilateral lesion). Finally, human cortical binding was investigated by autoradiography in three groups of subjects (control subjects with Braak ≤2, and AD patients, Braak >2 & ≤4 and Braak >4 stages).Results: We demonstrate that despite rapid peripheral metabolism of 18F-IAM6067, radiolabelled metabolites were hardly detected in brain samples. Brain uptake of 18F-IAM6067 showed differences in S1R anatomical distribution, namely from high to low uptake: pons-raphe, thalamus medio-dorsal, substantia nigra, hypothalamus, cerebellum, cortical areas and striatum. Pre-saturation studies showed 79-90% blockade of the binding in all areas of the brain indicated above except with the 5HT2B/C antagonist SB206553 and S2R ligand CM398 which induced no significant blockade, indicating good specificity of 18F-IAM6067 for S1Rs. No difference between ipsi- and contralateral sides of the brain in the mouse model of PD was detected. AMPA lesion induced a significant 69% decrease in 18F-IAM6067 uptake in the globus pallidus matching the neuronal loss as measured by NeuN, but only a trend to decrease (-16%) in the caudate putamen despite a significant 91% decrease in neuronal count. Moreover, no difference in the human cortical binding was shown between AD groups and controls.Conclusion: This work shows that 18F-IAM6067 is a specific and selective S1R radiotracer. The absence or small changes in S1R detected here in animal models and human tissue warrants further investigations and suggests that S1R might not be the anticipated ideal biomarker for neuronal loss in neurodegenerative diseases such as AD and PD.

MicroRNA-214 targets COX-2 to antagonize indoxyl sulfate (IS)-induced endothelial cell apoptosis.

Cardiovascular disease (CVD) serves as the major cause of mortality in chronic kidney disease (CKD) patients. The injury of endothelium associated with the long-term challenge of uremic toxins including the toxic indoxyl sulfate (IS) is one of key pathological factors leading to CVD. However, the mechanisms of uremic toxins, especially the IS, resulting in endothelial injury, remain unclear. miR-214 was reported to contribute to the pathogenesis of cardiovascular diseases, while its role in IS-induced endothelial cell apoptosis is unknown. In this study, we investigated the role of microRNA-214 (miR-214) in IS-induced endothelial cell apoptosis and the underlying mechanisms using mouse aortic endothelial cells (MAECs). Following IS treatment, miR-214 was significantly downregulated in MAECs in line with enhanced cell apoptosis. Meanwhile, COX-2 was upregulated at both mRNA and protein levels along with increased secretion of PGE2 in medium. To define the role of miR-214 in IS-induced endothelial cell apoptosis, we modulated miR-214 level in MAECs and found that overexpression of miR-214 markedly attenuated endothelial cell apoptosis, while antagonism of miR-214 deteriorated cell death after IS challenge. Further analyses confirmed that COX-2 is a target gene of miR-214, and the inhibition of COX-2 by a specific COX-2 inhibitor NS-398 strikingly attenuated IS-induced endothelial cell apoptosis along with a significant blockade of PGE2 secretion. In conclusion, this study demonstrated an important role of miR-214 in protecting against endothelial cell damage induced by IS possibly by direct downregulation of COX-2/PGE2 axis.

Abstract 2627: Inhibition of TGFβ in colorectal cancer cells is associated with a compensatory activation of AXL and p38 MAPK signaling pathways

Abstract According to the consensus molecular subtypes (CMS), almost 23% of human colorectal cancer (CRC) are classified in the CMS4 group, characterized by a mesenchymal signature and the activation of transforming growth factor β (TGF-β) signaling. AXL is a tyrosine kinase receptor involved in epithelial to mesenchymal transition, angiogenesis and immune modulation in CRC. We have previously demonstrated that AXL gene is amplified in approximately 5% of human CRC, and that AXL inhibition causes a significant blockade of CRC cell proliferation and migration. Here we have evaluated the role of TGF-β signaling and the potential interaction between TGF-β and AXL in human CRC cell lines. We assessed the expression and activation of TGF-β and AXL in a panel of six human CRC cell lines by western blot (WB) and real time PCR. We tested the sensitivity of HCT116 and LOVO cells to treatment with galunisertib ( LY21209761), a selective TGF-β R1 inhibitor, by MTT, cell invasion, wound healing and soft-agar colony forming assays. Further, intracellular CRC cell signaling was evaluated following galunisertib treatment by WB. TGF-β receptors 1 and 2 were expressed in all human CRC cell lines (HCT116, SW480, LOVO, LIM 1215, SW48 and CaCo2), whereas AXL expression was restricted to HCT116, SW480, LOVO cells. Treatment with galunisertib had little or no effect on cancer cell growth, whereas it partially reduced TGF-β induced cell migration, invasion and colony formation in HCT116 and LOVO cells (that co-expressed both TGF-β receptors and AXL). Interestingly, TGF-β inhibition resulted in a concomitant significant activation of AXL and p38 MAPK in both cell lines, that could represent a cancer cell adaptive mechanism of resistance to galunisertib treatment. These results suggest a potential functional cross talk between TGF-β, AXL and p38 MAPK signals in human CRC cells. In this respect, further experiments are ongoing to better understand this interaction with the aim of identifying potential novel anti-AXL and anti-TGF-β therapeutic strategies. Citation Format: Davide Ciardiello, Pietro Paolo Vitiello, Nunzia Matrone, Valentina Belli, Claudia Cardone, Luca Poliero, Carola Borrelli, Gianluca Arrichiello, Giulia Martini, Vincenza Ciaramella, Giusi Barra, Floriana Morgillo, Teresa Troiani, Davide Melisi, Fortunato Ciardiello, Erika Martinelli. Inhibition of TGFβ in colorectal cancer cells is associated with a compensatory activation of AXL and p38 MAPK signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2627.

Effects of dopaminergic and serotonergic compounds in rats trained to discriminate a high and a low training dose of the synthetic cathinone mephedrone.

The underlying pharmacological mechanisms of mephedrone, especially as related to interactions with different neurotransmitter systems, are a critical area of study as mephedrone continues to be abused. Direct-acting 5-HT2A/2C receptor agonists and antagonists and D1-3 receptor antagonists were examined in two groups of rats trained to discriminate mephedrone. A high dose of mephedrone was trained to extend previous results with traditional monoamine transporter inhibitors and substrate releasers. A very low dose of mephedrone was trained to preferentially capture serotonergic activity and to minimize the influence of rate-decreasing effects on substitution patterns. Selective 5-HT2A/2C and D1-3 receptor antagonists were examined in both groups. Male Sprague-Dawley rats were trained to discriminate either a low dose of 0.5mg/kg mephedrone (N = 24) or a high dose of 3.2mg/kg mephedrone (N = 11) from saline. In the low training-dose group, mephedrone, MDMA, methamphetamine, d-amphetamine, cocaine, and enantiomers of mephedrone substituted for mephedrone; mCPP partially substituted overall for mephedrone; and DOI, WAY163909, and morphine failed to substitute for mephedrone. In the high training-dose group, only mephedrone and MDMA substituted for mephedrone. Sulpiride produced a small antagonism of the low training dose of mephedrone while SCH23390, SB242084, and ketanserin altered response rates. A lower training dose of mephedrone produces a discriminative stimulus fully mimicked by MDMA, methamphetamine, cocaine, and d-amphetamine, whereas a higher training dose of mephedrone requires a discriminative stimulus that was only mimicked by MDMA. Dopaminergic or serotoninergic antagonists failed to produce significant blockade of mephedrone at either training dose.