The serotonin 5-HT7 receptor (5-HT7R), a member of the rhodopsin-like family of G protein-coupled receptors (GPCRs), is highly expressed in the central nervous system (CNS) and represents a promising target for treating CNS disorders such as sleep disturbances, migraine, neuropsychiatric conditions, and neuropathic pain. Because of its therapeutic potential, extensive efforts have focused on developing selective 5-HT7R ligands. In the last decades, biased signaling has emerged as a key concept in GPCR pharmacology as biased ligands can stabilize specific active states of the receptor and trigger selective activation of downstream signaling pathways. In this context, we recently identified two biased 5-HT7R ligands, Serodolin and MOA51, from different chemical series. Here, we aimed to compare the pharmacological and safety profiles of these ligands and assess their effect on pain-related behaviors and spinal neuroinflammation. In inflammatory pain models (acid acetic writhing, formalin, and CFA tests), both Serodolin and MOA51 effectively attenuated pain responses to a similar extent. Furthermore, in neuropathic pain models, spinal nerve injury (SNI) and the Cuff model, both ligands reversed mechanical allodynia. Interestingly, unlike pregabalin, a clinically used reference drug, neither Serodolin nor MOA51, induced apparent development of tolerance after 10 consecutive days of administration. Treatment with these 5-HT7R ligands also reduced spinal microglial activity and attenuated neuronal hyperactivity in the spinal cord. Altogether, these findings highlight the potential of 5-HT7R-biased ligands as promising analgesic candidates capable of modulating neuroinflammatory processes and mitigating both inflammatory and neuropathic pain.

Glioblastomas represent the most aggressive and lethal form of primary brain cancer and continue to pose a major challenge to global health. MicroRNAs (miRNAs), as central regulators of gene expression, are intimately involved in the initiation, progression, and therapeutic resistance of numerous malignancies, including glioblastoma. Therefore, this class of non-coding RNAs are considered to be valuable candidates for innovative therapeutic strategies. However, despite many promising preclinical studies, miRNA-based therapies have yet to be translated into routine clinical practice. In the context of glioblastoma, one of the principal obstacles to the effective delivery of synthetic miRNA therapy is their limited ability to cross the blood–brain barrier (BBB). To address this challenge, a variety of locoregional delivery strategies have been developed in recent years. In this review, we provide a detailed discussion and a state-of-the-art overview of these local delivery methods in the context of glioblastoma treatment, with a specific emphasis on their application for delivering miRNA-based therapeutic oligonucleotides, formulated either with or without synthetic nanoparticles.

MicroRNAs are short noncoding RNAs that play important roles in fine tuning genetic networks as genes post-transcriptional regulators. Monitoring the regulatory activity of microRNAs is technically challenging, especially in primary cells and 3-dimensional (3D) organotypic cultures. We optimized the previously reported RILES miRNA-ON sensor system to visualize the spatial expression of miR-203 and miR-30a by fluorescence imaging in 2-dimensional and 3D cultures of human primary keratinocytes. The generated system, called RIFES (RNAi-inducible fluorescence expression system), successfully imaged the expression of miR-30a-5p and miR-30a-3p in the suprabasal layers of the epidermis. This information was exploited to uncover the molecular mechanisms regulating the expression of miR-30a in human keratinocytes. We demonstrate that chemical inhibition of the Notch1 pathway induced GFP expression in undifferentiated RIFES/miR-30a keratinocyte cells, with fluorescence redistribution in the basal layers of 3D RIFES/miR-30a epidermis. Moreover, overexpressing miR-30a in 3D epidermal models resulted in NOTCH1 downregulation, suggesting a negative feedback loop between miR-30a and Notch. Because the Notch pathway was found downregulated in aged epidermis biopsies, we propose that Notch downregulation contributes to miR-30a induction during aging. Therefore, the RIFES system appears as a powerful tool to visualize the expression of microRNAs in 3D epidermis and to identify their potential upstream regulators.

Circulating microRNAs (miRNAs) have aroused a lot of interest as reliable blood diagnostic biomarkers of Alzheimer’s disease (AD). Here, we investigated the panel of expressed blood miRNAs in response to aggregated Aβ1–42 peptides infused in the hippocampus of adult rats to mimic events of the early onset of non-familial AD disorder. Aβ1–42 peptides in the hippocampus led to cognitive impairments associated with an astrogliosis and downregulation of circulating miRNA-146a-5p, -29a-3p, -29c-3p, -125b-5p, and-191-5p. We established the kinetics of expression of selected miRNAs and found differences with those detected in the APPswe/PS1dE9 transgenic mouse model. Of note, miRNA-146a-5p was exclusively dysregulated in the Aβ-induced AD model. The treatment of primary astrocytes with Aβ1–42 peptides led to miRNA-146a-5p upregulation though the activation of the NF-κB signaling pathway, which in turn downregulated IRAK-1 but not TRAF-6 expression. As a consequence, no induction of IL-1β, IL-6, or TNF-α was detected. Astrocytes treated with a miRNA-146-5p inhibitor rescued IRAK-1 and changed TRAF-6 steady-state levels that correlated with the induction of IL-6, IL-1β, and CXCL1 production, indicating that miRNA-146a-5p operates anti-inflammatory functions through a NF-κB pathway negative feedback loop. Overall, we report a panel of circulating miRNAs that correlated with Aβ1–42 peptides’ presence in the hippocampus and provide mechanistic insights into miRNA-146a-5p biological function in the development of the early stage of sporadic AD.

Supplementary Figure 1 from Cancer-Specific Transgene Expression Mediated by Systemic Injection of Nanoparticles

Supplementary Figure Legends 1-2 from Cancer-Specific Transgene Expression Mediated by Systemic Injection of Nanoparticles

Supplementary Figure 2 from Cancer-Specific Transgene Expression Mediated by Systemic Injection of Nanoparticles

Epidermal cells integrate multiple signals that activate the signaling pathways involved in skin homeostasis. TGF-β1 signaling pathway upregulates microRNA (miR)-21-5p in keratinocytes and is often deregulated in skin diseases. To identify the bioactive compounds that enable to modulate the TGF-β1/miR-21-5p signaling pathway, we screened a library of medicinal plant extracts using our miR-ON RILES luciferase reporter system placed under the control of the miR-21-5p in keratinocytes treated with TGF-β1. We identified silymarin, a mixture of flavonolignans extracted from Silybum marianum (L.) Gaertn., as the most potent regulator of miR-21-5p expression. Using Argonaute 2 immunoprecipitation and RT-qPCR, we showed that silymarin regulates the expression of miR-21-5p through a noncanonical TGF-β1 signaling pathway, whereas RNA-sequencing analysis revealed three unexpected transcriptomic signatures associated with keratinocyte differentiation, cell cycle, and lipid metabolism. Mechanistically, we demonstrated that SM blocks cell cycle progression, inhibits keratinocyte differentiation through repression of Notch3 expression, stimulates lipid synthesis via activation of PPARγ signaling and inhibits inflammatory responses by suppressing the transcriptional activity of NF-κB. We finally showed that topical application of silymarin alleviates the development of imiquimod-induced psoriasiform lesions in mice by abrogating the altered expression levels of markers involved in inflammation, proliferation, differentiation, and lipid metabolism.

A major unresolved challenge in miRNA biology is the capacity to monitor the spatiotemporal activity of miRNAs expressed in animal disease models. We recently reported that the miRNA-ON monitoring system called RILES (RNAi-inducible expression Luciferase system) implanted in lentivirus expression system (LentiRILES) offers unique opportunity to decipher the kinetics of miRNA activity in vitro, in relation with their intracellular trafficking in glioblastoma cells. In this study, we describe in detail the method for the production of LentiRILES stable cell lines and employed it in several applications in the field of miRNA biology and therapy. We show that LentiRILES is a robust, highly specific and sensitive miRNA sensor system that can be used in vitro as a single-cell miRNA monitoring method, cell-based screening platform for miRNA therapeutics and as a tool to analyse the structure-function relationship of the miRNA duplex. Furthermore, we report the kinetics of miRNA activity upon the intracranial delivery of miRNA mimics in an orthotopic animal model of glioblastoma. This information is exploited to evaluate the tumour suppressive function of miRNA-200c as locoregional therapeutic modality to treat glioblastoma. Our data provide evidence that LentiRILES is a robust system, well suited to resolve the activity of endogenous and exogenously expressed miRNAs from basic research to gene and cell therapy.

Psoriasis is a chronic inflammatory skin disease that is mediated by complex crosstalk between immune cells and keratinocytes (KCs). Emerging studies have showed a specific psoriatic microRNAs signature, in which miR-21 is one of the most upregulated and dynamic miRNAs. In this study, we focused our investigations on the passenger miR-21-3p strand, which is poorly studied in skin and in psoriasis pathogenesis. Here, we showed the upregulation of miR-21-3p in an IMQ-induced psoriasiform mouse model. This upregulation was correlated with IL-22 expression and functionality, both in vitro and in vivo, and it occurred via STAT3 and NF-κB signaling. We identified a network of differentially expressed genes involved in abnormal proliferation control and immune regulatory genes implicated in the molecular pathogenesis of psoriasis in response to miR-21-3p overexpression in KCs. These results were confirmed by functional assays that validated the proliferative potential of miR-21-3p. All these findings highlight the importance of miR-21-3p, an underestimated miRNA, in psoriasis and provide novel molecular targets for therapeutic purposes.

In order to harness local resources to improve well-being and human health, we aim in this study to investigate if the microalgae Dunaliella sp. isolated from the Tunisian coastal zone possesses any anticancer activity. Dunaliella sp. was cultured under normal (DSC) or stressed (DSS) conditions and extracted using different procedures. The biological activity assessment was performed on the Triple Negative Breast Cancer (TNBC) using 4T1 murine cells as a model. Results indicate that: (i) aqueous extract was the most cytotoxic compared to ethanolic and hydroalcoholic extracts; (ii) DSS activity was superior to that of DSC. DSS extracts induced apoptosis rather than necrosis, as evidenced by DNA fragmentation, PARP-1 cleavage and caspase-3 activation. Evaluation in an orthotopic TNBC model validated the anticancer activity in vivo. Intratumoral injection of DSS extract resulted in reduced tumor growth and an enhanced immune system activation. On the transcriptional side, the expression level of the immunosuppressive enzyme Arg-1 was decreased, as well as those of NOS-2 and COX-2 genes. These results suggest a potential anticancer activity of Tunisian Dunaliella sp. deserving further attention.

Insect venom is a highly complex mixture of bioactive compounds, containing proteins, peptides, and small molecules. Environmental factors can alter the venom composition and lead to intraspecific variation in its bioactivity properties. The investigation of discriminating compounds caused by variation impacts can be a key to manage sampling and explore the bioactive compounds. The present study reports the development of a peptidomic methodology based on UHPLC–ESI-QTOF–HRMS analysis followed by a nontargeted multivariate analysis to reveal the profile variance of Vespa velutina venom collected in different conditions. The reliability of the approach was enhanced by optimizing certain XCMS data processing parameters and determining the sample peak threshold to eliminate the interfering features. This approach demonstrated a good repeatability and a criterion coefficient of variation (CV) > 30% was set for deleting nonrepeatable features from the matrix. The methodology was then applied to investigate the impact of collection period variation. PCA and PLS-DA models were used and validated by cross-validation and permutation tests. A slight discrimination was found between winter and summer hornet venom in two successive years with 10 common discriminating compounds.

MicroRNA (miRNA) oligonucleotides therapeutics are potent and attractive drugs for cancer treatment, but the kinetics of their intracellular trafficking, RISC processing and interaction with their mRNA targets in the cells are still not well understood. Moreover, the absence of efficient carriers impairs their translation into the clinic. Here, we compare the kinetics of miRNA-133a activity after transfection of U87MG glioblastoma cells with either a home-made lipopolyplexes (LPRi) or with the RNAiMax transfection reagent. For this purpose, we combined miRNA intracellular trafficking studies by confocal microscopy with our previously described RILES miRNA-ON reporter system subcloned here in a lentivirus expression vector (LentiRILES) for longitudinal analysis of miRNA activity in transfected cells. Using the LentiRILES system, we report significant differences in terms of miRNA delivery kinetics performed by these two transfection regents. We decipher the mechanisms of miRNA delivery by LPRi and investigate the main steps of miRNA internalization and cytosolic processing. We demonstrate that LPRi preferentially uses caveolae-mediated endocytosis as the main internalization pathway, releases miRNA into the cytosol after the first 3 h of incubation, and addresses the cytosolic miRNAs to P-bodies, while a fraction of miRNAs are exported to the extracellular space through exosomes which were found fully capable to re-transfect the cells. We implanted the LentiRILES cells in the brain of mice and infused the tumours with LPRi.miRNA using the convection-enhanced delivery method. Bioluminescence imaging of the live mice revealed efficient delivery of miRNAs in glioblastoma tumours, attesting successful miRNA uptake, internalization and RISC activation in vivo. Overall, our study provides a comprehensive overview of miRNA intracellular trafficking and processing in a glioblastoma context and highlights the potential use of LPRi for miRNA-based therapy.

The present study aimed to explore the potential antioxidant molecules of the Asian hornet venom (Vespa velutina nigrithorax) responsible for radical scavenging activity and human keratinocyte protection against oxidative stress. We developed a first technical platform that combined a DPPH radical scavenging chemical assay and cytotoxicity and ROS (reactive oxygen species) production in HaCaT keratinocyte cells exposed to UVB to evaluate the antioxidant property of V. velutina venom. We further employed Thin Layer Chromatography (TLC) combined with the DPPH assay as a targeted separation approach to isolate the antioxidant compounds responsible for the free radical scavenging property of V. velutina venom. In parallel, the latter was fractionated by a HPLC-DAD non-targeted separation approach. From this experiment, nine fractions were generated which were again evaluated separately for their antioxidant properties using DPPH assays. Results showed that only one fraction exhibited significant antioxidant activity in which serotonin was identified as the major compound by a UHPLC-ESI-QTOF HRMS/MS approach. We finally demonstrated, using purified serotonin molecule that this bioactive structure is mostly responsible for the free radical scavenging property of the crude venom as evidenced by DPPH and ROS assays in HaCaT cells exposed to UVB.

The potential reprotoxicity of bifenthrin remains unclear if only the common clinical indicators of reproductive disease are examined. The present study aimed to investigate the efficacy of Spirulina platensis, a microalga rich in antioxidant compounds, against bifenthrin-induced testicular oxidative damage in male mice. At the first, we demonstrate that administration of bifenthrin resulted in a decline of testosterone level and in deterioration of sperm quality that was correlated with significant transcription changes of some specific mRNA and microRNA involved in cholesterol transport, testosterone synthesis, and spermatogenesis. At the biochemical level, we found that oxidative stress was obvious in the bifenthrin group, as evidenced by increase in malondialdehyde (MDA), protein carbonyls (PCO), reactive oxygen species (ROS), and nitrite oxide (NO) that was correlated with activation of genes related to mitochondrial apoptotic signal pathways. We then brought, for the first time to our knowledge, solid and complete experimental evidences that administration of mice with Spirulina extract was sufficient to protect against deleterious effects BF in testicular tissues by abrogating the change in antioxidant enzyme activities; the increase in MDA, PCO, and NO concentrations; and the altered expression level of miRNA and mRNA involved in spermatogenesis. We finally demonstrate that Spirulina restores the production of testosterone in mice as well as epididymal sperm viability and motility. These results suggest a potential antitoxic activity of Tunisian Spirulina deserving further attention.

Dunaliella is a green microalga known for its ability to produce high levels of carotenoids under well-defined growing conditions. Molecular responses to the simultaneous effect of increasing salinity, light intensity and decrease of nitrogen availability were investigated in terms of their effect on different metabolic pathways (isoprenoids synthesis, glycolysis, carbohydrate use, etc.) by following the transcriptional regulation of enolase (ENO), 1-deoxy-D-xylulose 5-phosphate synthase (DXS), lycopene β-cyclase (LCYB), carotene globule protein (CGP), chloroplast-localized heat shock protein (HSP70), and chloroplast ribulose phosphate-3-epimerase (RPE) genes. The intracellular production of carotenoid was increased five times in stressed Dunaliella cells compared to those grown in an unstressed condition. At transcriptional levels, ENO implicated in glycolysis, and revealing about polysaccharides degradation, showed a two-stage response during the first 72 h. Genes directly involved in β-carotene accumulation, namely, CGP and LCYB, revealed the most important increase by about 54 and 10 folds, respectively. In silico sequence analysis, along with 3D modeling studies, were performed to identify possible posttranslational modifications of CGP and LCYB proteins. Our results described, for the first time, their probable regulation by sumoylation covalent attachment as well as the presence of expressed SUMO (small ubiquitin-related modifier) protein in Dunaliella sp.

miRNAs are key cell regulatory elements code by the genome. Although their signature in cancer is incompletely known and not yet an element of diagnosis, the role of oncomiRs become increasingly documented. Alongside cancer intrinsic signals in which they exert essential functions, miRNAs are also responding to environmental cues. Developing novel approaches to target miRNA-pathways represents a pivotal issue in research and may result in a major breakthrough for human health. Due to their submicron size and versatile physicochemical properties the emergence of innovative nanomedicines may be here largely advantageous for the development of targeted clinical strategies. With a special focus on glioblastoma, the present review addresses cancer functions of miRNAs and raises the question of an adapted nanomedicine.

The editors were made aware of several concerns pertaining to Figs. 1, 2, and 4 in [this article][1] ([1][2]). The Western blot images in Figs. 1 and 4 were prepared based on the acceptable standards at the time of submission. In Fig. 2C, duplicate migration/invasion assay images were used to

MicroRNAs (miRNAs) are key regulatory elements encoded by the genome. A single miRNA can downregulate the expression of multiple genes involved in diverse functions. Because cancer is a disease with multiple gene aberrations, developing novel approaches to identify and modulate miRNA pathways may result in a breakthrough for cancer treatment. With a special focus on glioblastoma (GBM), this review provides an up-to-date summary of miRNA biogenesis, the role of miRNA in cancer resistance, and essential tools for modulating miRNA expression, as well as of clinically promising RNAi delivery systems and how they can be adapted for therapy.