Namodenoson Inhibits the Growth of Pancreatic Carcinoma via Deregulation of the Wnt/ß-catenin, NF-κB, and RAS Signaling Pathways.

Namodenoson, an A3 adenosine receptor (A3AR) agonist, is currently being used in a phase III trial in advanced liver cancer. We examined the anti-growth effect of namodenoson on pancreatic carcinoma cells and investigated the molecular mechanism involved. BxPC-3 pancreatic carcinoma cells were cultured with namodenoson (5-20 nM for 24 h at 37 °C), and the Presto Blue assay was used to monitor cell growth. Western blot analyses were performed on BxPC-3 cells (20 nM namodenoson for 24 h at 37 °C) to evaluate the expression levels of cell growth regulatory proteins. In vivo studies involved the subcutaneous inoculation of BxPC-3 cells into nude mice, randomizing the mice into namodenoson (10 µg/kg twice daily for 35 days) vs. control, and monitoring tumor size twice weekly. Treatment with namodenoson was associated with the significant dose-dependent inhibition of BxPC-3 cell growth, which was mitigated by the A3AR antagonist MRS1523. Western blot analyses showed that namodenoson treatment modulated the expression of NF-κB, as well as proteins in the Wnt/ß-catenin and the RAS signaling pathways, leading to the upregulation of apoptotic proteins (Bad, Bax). In vivo studies also showed the significant inhibition of pancreatic carcinoma tumor growth with namodenoson. In conclusion, our findings support the continued development of namodenoson as a treatment for pancreatic cancer.

Using co-creation focus groups to customise a remote multidomain programme designed to increase dementia literacy.

OBJECTIVES: To adapt the content and functionalities of Brain Health PRO, a web-based multidomain program designed to increase dementia literacy, to the context and needs of users, providers and community organisations across Québec, Canada. DESIGN: Five consecutive qualitative co-creation focus group sessions 30-90 min in duration each, exploring potential barriers and facilitators to usability, accessibility, comprehensibility, participant recruitment and retention. SETTING: Virtual meetings. PARTICIPANTS: A 15-member team based in Québec and Ontario, Canada, consisting of 9 researchers (including a graduate student and the project coordinator), representing occupational therapy, sensory rehabilitation, neuropsychology, psychology, health science and research methods, 3 informal caregivers of older adults living with cognitive decline and 3 members of the Federation of Quebec Alzheimer Societies. DATA ANALYSIS: Session recordings were summarised through both qualitative description and thematic analysis. RESULTS: The synthesised recommendations included adjustments around diversity, the complexity and presentation styles of the materials, suggestions on refining the web interface and the measurement approaches; it influenced aspects of participant recruitment, retention efforts and engagement with the content of Brain Health PRO. CONCLUSIONS: Co-creation in dementia prevention research is important because it involves collaboration between researchers, community support and service providers, and persons with lived experience as care providers, in the design and implementation of clinical studies. This approach helps to ensure that the content and presentation of educational material is relevant and meaningful to the target population and those involved in its delivery, and it leads to a greater understanding of their needs and perspectives.

A3 adenosine receptor allosteric modulator CF602 reverses erectile dysfunction in a diabetic rat model.

Adenosine plays a major role in erection by binding to its receptors and activating pathways resulting in increased arterial blood flow and intracavernosal pressure (ICP). CF602, an allosteric modulator of the A3 adenosine receptor (A3AR), increases the binding affinity of the endogenous adenosine to the receptor. We examined the effect of CF602 on resolving erectile dysfunction (ED) in a diabetic ED rat model (streptozotocin-induced diabetic rats that were screened for ED using the apomorphine test). ED was assessed by measuring ICP and main arterial pressure (MAP) during electrostimulation of the cavernosal nerve. A single dose of CF602 or placebo was applied either topically (100 µl from a 100 nM or 500 nM solution) or orally (100, 200 or 500 µg/kg) prior to erectile function assessment. A significant dose-dependent improvement in the ICP:MAP ratio without a change in MAP was recorded with the topical and oral CF602 treatments. A significant increase in smooth muscle:collagen ratio, vascular endothelial growth factor and endothelial nitric oxide synthase was also observed in both administration modes. In conclusion, topical and oral treatment with CF602 significantly improved erectile function, supporting its further evaluation as a treatment for ED.

Randomised clinical trial: A phase 2 double-blind study of namodenoson in non-alcoholic fatty liver disease and steatohepatitis.

BACKGROUND: Namodenoson, an A3 adenosine receptor (A3AR) agonist, improved liver function/pathology in non-alcoholic steatohepatitis (NASH) preclinical models. AIM: To evaluate the efficacy and safety of namodenoson for the treatment of non-alcoholic fatty liver disease (NAFLD) with or without NASH METHODS: This phase 2 study included 60 patients with NAFLD (ALT ≥60 IU/L) who were randomised (1:1:1) to oral namodenoson 12.5 mg b.d. (n = 21), 25 mg b.d. (n = 19), or placebo (n = 20) for 12 weeks (total follow-up: 16 weeks). The main efficacy endpoint involved serum ALT after 12 weeks of treatment. RESULTS: Serum ALT decreased over time with namodenoson in a dose-dependent manner. The difference between change from baseline (CFB) for ALT in the namodenoson 25 mg b.d. arm vs placebo trended towards significance at 12 weeks (P = 0.066). Serum AST levels also decreased with namodenoson in a dose-dependent manner; at 12 weeks, the CFB for 25 mg b.d. vs placebo was significant (P = 0.03). At Week 12, 31.6% in the namodenoson 25 mg b.d. arm and 20.0% in the placebo arm achieved ALT normalisation (P = 0.405). At week 16, the respective rates were 36.8% and 10.0% (P = 0.038). A3AR expression levels were stable over time across study arms. Both doses of namodenoson were well tolerated with no drug-emergent severe adverse events, drug-drug interactions, hepatotoxicity, or deaths. Three adverse events were considered possibly related to study treatment: myalgia (12.5 mg b.d. arm), muscular weakness (25 mg b.d. arm), and headache (25 mg b.d. arm). CONCLUSION: A3AR is a valid target; namodenoson 25 mg b.d. was safe and demonstrated efficacy signals (ClinicalTrials.gov #NCT02927314).

Namodenoson in Advanced Hepatocellular Carcinoma and Child-Pugh B Cirrhosis: Randomized Placebo-Controlled Clinical Trial.

Namodenoson, an A3 adenosine-receptor agonist, showed promising results in advanced hepatocellular carcinoma (HCC) and moderate hepatic dysfunction (Child-Pugh B; CPB) in a phase I/II clinical study. This phase II study investigated namodenoson as second-line therapy in such patients. Patients were randomized 2:1 to twice a day (BID) namodenoson (25 mg; n = 50) or placebo (n = 28). The primary endpoint (overall survival [OS]) was not met. Median OS was 4.1/4.3 months for namodenoson/placebo (hazard ratio [HR], 0.82; 95% confidence interval [CI] 0.49-1.38; p = 0.46). Pre-planned subgroup analysis of CPB7 patients (34 namodenoson-treated, 22 placebo-treated) showed a nonsignificant improvement in OS/progression-free survival (PFS). OS: 6.9 versus 4.3 months; HR, 0.81; 95% CI: 0.45-1.43, p = 0.46. PFS: 3.5 versus 1.9 months; HR, 0.89; 95% CI: 0.51-1.55, p = 0.67 (log-rank test). The difference in 12-month OS was significant (44% versus 18%, p = 0.028). Response rates were determined in patients for whom ≥ 1 assessment post-baseline was available (34 namodenoson-treated, 21 placebo-treated). Partial response was achieved by 3/34 (8.8%) and 0/21 (0%) patients, respectively. Namodenoson was well-tolerated, with a safety profile comparable to that of the placebo group. No treatment-related deaths were reported; no patients withdrew due to toxicity. In conclusion, namodenoson demonstrated a favorable safety profile and a preliminary efficacy signal in HCC CPB.

The A3 adenosine receptor agonist, namodenoson, ameliorates non­alcoholic steatohepatitis in mice.

The Wnt/ß­catenin pathway confers a chain of molecular events in livers affected by non­alcoholic steatohepatitis (NASH). Namodenoson, a selective agonist of the A3 adenosine receptor (A3AR), which is highly expressed in pathological liver cells, induces a robust anti­inflammatory effect in the liver, mediated via the de­regulation of the Wnt/ß­catenin pathway. Namodenoson also acts as a liver protective agent by inhibiting ischemia/reperfusion injury. Based on these unique characteristics, we investigated the anti­NASH effect of Namodenoson in murine models of steatohepatitis and in the LX2 human hepatic stellate cell line (HSC). In the STAM model, Namodenoson significantly decreased the non­alcoholic fatty liver disease (NAFLD) activity score, NAS, demonstrating anti­inflammatory and anti­steatotic effects. In the carbon tetrachloride (CCl4) model, Namodenoson reversed alanine aminotransferase (ALT) to normal values and significantly improved liver inflammation and fibrosis, as well as the adiponectin and leptin levels. Namodenoson de­regulated the Wnt/ß­catenin pathway in the liver extracts of the CCl4 model mice and in the LX2 HSCs, manifested by a decrease in the expression of phosphoinositide 3­kinase (PI3K), nuclear factor κ­light­chain­enhancer of activated B cells (NF­κB), ß­catenin, lymphoid enhancer­binding factor 1 (Lef­1) and cyclin D1, and an increase in the expression level of glycogen synthase kinase 3ß (GSK­3ß). The fibrosis marker, α­smooth muscle actin (α­SMA) was also de­regulated, supporting the anti­fibrotic effect of Namodenoson. On the whole, the findings of this study demonstrate that Namodenoson exerts an anti­NASH effect mediated via the de­regulation of the PI3K/NF­κB/Wnt/ß­catenin signaling pathway. Thus, targeting A3AR may prove to be a novel direction in the pharmacotherapy of NAFLD/NASH.

Adaptive and selective production of syllable duration and fundamental frequency as word segmentation cues by French-English bilinguals.

Despite the significant impact of prosody on L2 speakers' intelligibility, few studies have examined the production of prosodic cues associated with word segmentation in non-native or non-dominant languages. Here, 62 French-English bilingual adults, who varied in L1 (French or English) and language dominance, produced sentences built around syllable strings that can be produced either as one bisyllabic word or two monosyllabic words. Each participant produced both English and French utterances, providing both native productions (used as reference) and L2 productions. Acoustic analyses of the mean fundamental frequency (F0) and duration of both syllables of the ambiguous string revealed that speakers' relative language dominance affected the speakers' prosodic cue production over and above L1. Speakers also produced different prosodic patterns in English and French, suggesting that the production of prosodic cues associated with word-segmentation is both adaptive (modified by language experience) and selective (specific to each language).

Inhibition of IL-17 and IL-23 in Human Keratinocytes by the A3 Adenosine Receptor Agonist Piclidenoson.

Interleukin-17 and interleukin-23 play major roles in the inflammatory process in psoriasis. The Gi protein-associated A3 adenosine receptor (A3AR) is known to be overexpressed in inflammatory cells and in peripheral blood mononuclear cells (PBMCs) of patients with autoimmune inflammatory conditions. Piclidenoson, a selective agonist at the A3AR, induces robust anti-inflammatory effect in psoriasis patients. In this study, we aimed to explore A3AR expression levels in psoriasis patients and its role in mediating the anti-inflammatory effect of piclidenoson in human keratinocyte cells. A3AR expression levels were evaluated in skin tissue and PBMCs derived from psoriasis patients and healthy subjects. Proliferation assay and the expression of signaling proteins were used to evaluate piclidenoson effect on human keratinocytes (HaCat). High A3AR expression levels were found in a skin biopsy and in PBMCs from psoriasis patients in comparison to healthy subjects. Piclidenoson inhibited the proliferation of HaCat cells through deregulation of the NF-κB signaling pathway, leading to a decrease in interleukin-17 and interleukin-23 expression levels. This effect was counteracted by the specific antagonist MRS 1523. A3AR overexpression in skin and PBMCs of psoriasis patients may be used as a target to inhibit pathological cell proliferation and the production of interleukin-17 and interleukin-23.

The Kaposi's-sarcoma-associated herpesvirus orf35 gene product is required for efficient lytic virus reactivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) is implicated in the etiology of several human malignancies. KSHV open reading frame (orf) 35 encodes a conserved gammaherpesvirus protein with an, as yet, unknown function. Employing the bacterial artificial chromosome (BAC) system, we generated a recombinant viral clone that fails to express ORF35 (BAC16-ORF35-stop) but preserves intact adjacent and overlapping reading frames. Using this construct, we studied the role of this previously uncharacterized gene product during lytic reactivation of KSHV. Upon lytic reactivation, the ORF35-stop recombinant virus displayed significantly reduced lytic viral gene expression, viral DNA replication, and progeny virus production as compared to control wild-type virus. Exogenous expression of ORF35-Flag reversed the effects of ORF35 deficiency. These results demonstrate that ORF35 is important for efficient lytic virus reactivation.

Misleading Bias-Driven Expectations in Referential Processing and the Facilitative Role of Contrastive Accent.

Probabilistic preferences are often facilitative in language processing and may assist in discourse prediction. However, occasionally these sources of information may lead to inaccurate expectations. The current study investigated a test case of this scenario. An eye-tracking experiment examined the interpretation of ambiguous personal pronouns in the context of implicit causality biases. We tested whether reference resolution may be facilitated online by contrastive accent in cases of a bias-inconsistent referent. Implicit causality biases directed looks to the biased noun phrase; however, when the name of the bias-inconsistent antecedent was accented (e.g., JOHN envied Bill because he ...), this tendency was modulated. Contrastive accent seems to dampen the occasionally confusing prediction of implicit causality biases in referential processing. This demonstrates one way in which the spoken language comprehension system copes with occasional misguidance of otherwise helpful probabilistic information.

Fluorescent tagging and cellular distribution of the Kaposi's sarcoma-associated herpesvirus ORF45 tegument protein.

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is a cancer-related human virus, classified as a member of the Gammaherpesvirinae subfamily. We report here the construction of a dual fluorescent-tagged KSHV genome (BAC16-mCherry-ORF45), which constitutively expresses green fluorescent protein (GFP) and contains the tegument multifunctional ORF45 protein as a fusion protein with monomeric Cherry fluorescent protein (mCherry). We confirmed that this virus is properly expressed and correctly replicates and that the mCherry-ORF45 protein is incorporated into the virions. Using this labeled virus, we describe the dynamics of mCherry-ORF45 expression and localization in newly infected cells as well as in latently infected cells undergoing lytic induction and show that mCherry can be used to monitor cells undergoing the lytic viral cycle. This virus is likely to enable future studies monitoring the dynamics of viral trafficking and tegumentation during viral ingress and egress. IMPORTANCE: The present study describes the construction and characterization of a new recombinant KSHV genome BAC16 clone which expresses mCherry-tagged ORF45. This virus enables the tracking of cells undergoing lytic infection and can be used to address issues related to the trafficking and maturation pathways of KSHV virions.

Effects of cooperating and conflicting prosody in spoken English garden path sentences: ERP evidence for the boundary deletion hypothesis.

In reading, a comma in the wrong place can cause more severe misunderstandings than the lack of a required comma. Here, we used ERPs to demonstrate that a similar effect holds for prosodic boundaries in spoken language. Participants judged the acceptability of temporarily ambiguous English "garden path" sentences whose prosodic boundaries were either in line or in conflict with the actual syntactic structure. Sentences with incongruent boundaries were accepted less than those with missing boundaries and elicited a stronger on-line brain response in ERPs (N400/P600 components). Our results support the notion that mentally deleting an overt prosodic boundary is more costly than postulating a new one and extend previous findings, suggesting an immediate role of prosody in sentence comprehension. Importantly, our study also provides new details on the profile and temporal dynamics of the closure positive shift (CPS), an ERP component assumed to reflect prosodic phrasing in speech and music in real time. We show that the CPS is reliably elicited at the onset of prosodic boundaries in English sentences and is preceded by negative components. Its early onset distinguishes the speech CPS in adults both from prosodic ERP correlates in infants and from the "music CPS" previously reported for trained musicians.

Prosody-syntax interactions in aging: event-related potentials reveal dissociations between on-line and off-line measures.

This study used ERPs to determine whether older adults use prosody in resolving early and late closure ambiguities comparably to young adults. Participants made off-line acceptability judgments on well-formed sentences or those containing prosody-syntax mismatches. Behaviorally, both groups identified mismatches, but older subjects accepted mismatches significantly more often than younger participants. ERP results demonstrate CPS components and garden-path effects (P600s) in both groups, however, older adults displayed no N400 and more anterior P600 components. The data provide the first electrophysiological evidence suggesting that older adults process and integrate prosodic information in real-time, despite off-line behavioral differences. Age-related differences in neurocognitive processing mechanisms likely contribute to this dissociation.

Event-related potentials show online influence of lexical biases on prosodic processing.

This event-related potential study examined how the human brain integrates (i) structural preferences, (ii) lexical biases, and (iii) prosodic information when listeners encounter ambiguous 'garden path' sentences. Data showed that in the absence of overt prosodic boundaries, verb-intrinsic transitivity biases influence parsing preferences (late closure) online, resulting in a larger P600 garden path effect for transitive than intransitive verbs. Surprisingly, this lexical effect was mediated by prosodic processing, a closure positive shift brain response was elicited in total absence of acoustic boundary markers for transitively biased sentences only. Our results suggest early interactive integration of hierarchically organized processes rather than purely independent effects of lexical and prosodic information. As a primacy of prosody would predict, overt speech boundaries overrode both structural preferences and transitivity biases.