Generation and characterization of CRISPR-Cas9-mediated XPC gene knockout in human skin cells.

Xeroderma pigmentosum group C (XPC) is a versatile protein crucial for sensing DNA damage in the global genome nucleotide excision repair (GG-NER) pathway. This pathway is vital for mammalian cells, acting as their essential approach for repairing DNA lesions stemming from interactions with environmental factors, such as exposure to ultraviolet (UV) radiation from the sun. Loss-of-function mutations in the XPC gene confer a photosensitive phenotype in XP-C patients, resulting in the accumulation of unrepaired UV-induced DNA damage.

Synthetic rescue of Xeroderma Pigmentosum C phenotype via PIK3C3 downregulation.

Xeroderma Pigmentosum C is a dermal hereditary disease caused by a mutation in the DNA damage recognition protein XPC that belongs to the Nucleotide excision repair pathway. XPC patients display heightened sensitivity to light and an inability to mend DNA damage caused by UV radiation, resulting in the accumulation of lesions that can transform into mutations and eventually lead to cancer. To address this issue, we conducted a screening of siRNAs targeting human kinases, given their involvement in various DNA repair pathways, aiming to restore normal cellular behavior.

CRISPR-Cas9 Technology for the Creation of Biological Avatars Capable of Modeling and Treating Pathologies: From Discovery to the Latest Improvements.

This is a spectacular moment for genetics to evolve in genome editing, which encompasses the precise alteration of the cellular DNA sequences within various species. One of the most fascinating genome-editing technologies currently available is Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and its associated protein 9 (CRISPR-Cas9), which have integrated deeply into the research field within a short period due to its effectiveness. It became a standard tool utilized in a broad spectrum of biological and therapeutic applications.

A Bayesian Implementation of Quality-by-Design for the Development of Cationic Nano-Lipid for siRNA Transfection.

Unlike Quality by Testing approach, where products were tested only after drug manufacturing, Quality by Design (QbD) is a proactive control quality paradigm, which handles risks from the early development steps. In QbD, regression models built from experimental data are used to predict a risk mapping called Design Space in which the developers can identify values of critical input factors leading to acceptable probabilities to meet the efficacy and safety specifications for the expected product. These empirical models are often limited to quantitative responses.

Isoconazole and Clemizole Hydrochloride Partially Reverse the Xeroderma Pigmentosum C Phenotype.

Xeroderma Pigmentosum protein C (XPC) is involved in recognition and repair of bulky DNA damage such as lesions induced by Ultra Violet (UV) radiation. -mutated cells are, therefore, photosensitive and accumulate UVB-induced pyrimidine dimers leading to increased cancer incidence. Here, we performed a high-throughput screen to identify chemicals capable of normalizing the XP-C phenotype (hyper-photosensitivity and accumulation of photoproducts).

Therapeutic siRNAs Targeting the JAK/STAT Signalling Pathway in Inflammatory Bowel Diseases.

Background And Aims: Inflammatory bowel diseases are highly debilitating conditions that require constant monitoring and life-long medication. Current treatments are focused on systemic administration of immunomodulatory drugs, but they have a broad range of undesirable side-effects. RNA interference is a highly specific endogenous mechanism that regulates the expression of the gene at the transcript level, which can be repurposed using exogenous short interfering RNA [siRNA] to repress expression of the target gene.

High-throughput synthetic rescue for exhaustive characterization of suppressor mutations in human genes.

Inherited or acquired mutations can lead to pathological outcomes. However, in a process defined as synthetic rescue, phenotypic outcome created by primary mutation is alleviated by suppressor mutations. An exhaustive characterization of these mutations in humans is extremely valuable to better comprehend why patients carrying the same detrimental mutation exhibit different pathological outcomes or different responses to treatment.

Moderate hypothermia inhibits both proliferation and migration of human glioblastoma cells.

Purpose: Glioblastoma is the most aggressive malignant brain tumor. Despite multimodal treatments, median survival is only 15 months for glioblastoma patients, with tumor recurring in the resection margins after surgical removal. Hypothermia is emerging as an interesting and safe treatment for several conditions.

Androgen receptor-modulatory microRNAs provide insight into therapy resistance and therapeutic targets in advanced prostate cancer.

Androgen receptor (AR) signalling is a key prostate cancer (PC) driver, even in advanced 'castrate-resistant' disease (CRPC). To systematically identify microRNAs (miRs) modulating AR activity in lethal disease, hormone-responsive and -resistant PC cells expressing a luciferase-based AR reporter were transfected with a miR inhibitor library; 78 inhibitors significantly altered AR activity. Upon validation, miR-346, miR-361-3p and miR-197 inhibitors markedly reduced AR transcriptional activity, mRNA and protein levels, increased apoptosis, reduced proliferation, repressed EMT, and inhibited PC migration and invasion, demonstrating additive effects with AR inhibition.

MYPT1 is targeted by miR-145 inhibiting viability, migration and invasion in 2D and 3D HeLa cultures.

The miR-143/145 cluster is down-regulated in cervical tumor cells suggesting a role in tumorigenesis including cytoskeleton remodeling, a key event for tumor progression. The aim of the present work was to determine the role of miR-143/145 in the modulation of the myosin regulator phospho-myosin light chain (pMLC). HeLa monolayer and tridimensional cultures were transfected with miR-143 or miR-145 mimics inhibiting cell viability, proliferation, migration and invasion, mainly through miR-145.

Distinct outcomes of CRL-Nedd8 pathway inhibition reveal cancer cell plasticity.

Inhibition of protein degradation by blocking Cullin-RING E3 ligases (CRLs) is a new approach in cancer therapy though of unknown risk because CRL inhibition may stabilize both oncoproteins and tumor suppressors. Probing CRLs in prostate cancer cells revealed a remarkable plasticity of cells with TMPRSS2-ERG translocation. CRL suppression by chemical inhibition or knockdown of RING component RBX1 led to reversible G0/G1 cell cycle arrest that prevented cell apoptosis.

Φ-score: A cell-to-cell phenotypic scoring method for sensitive and selective hit discovery in cell-based assays.

Phenotypic screening monitors phenotypic changes induced by perturbations, including those generated by drugs or RNA interference. Currently-used methods for scoring screen hits have proven to be problematic, particularly when applied to physiologically relevant conditions such as low cell numbers or inefficient transfection. Here, we describe the Φ-score, which is a novel scoring method for the identification of phenotypic modifiers or hits in cell-based screens.

A statistically inferred microRNA network identifies breast cancer target miR-940 as an actin cytoskeleton regulator.

MiRNAs are key regulators of gene expression. By binding to many genes, they create a complex network of gene co-regulation. Here, using a network-based approach, we identified miRNA hub groups by their close connections and common targets.

The modulation of attachment, growth and morphology of cancerous prostate cells by polyelectrolyte nanofilms.

The behaviour of cancerous epithelial prostatic cells (PC3) growing on polyelectrolytes (PE) coatings was compared to the behaviour of immortalized normal prostatic cells (PNT-2). The cell behaviour was evaluated and quantified in terms of initial cell attachment, growth, metabolic activity, morphometry, adhesion, apoptosis and stress related gene expression. Both the anionic PSS (poly(sodium 4-styrenesulphonate))-terminated surface and cationic PAH (poly(allylamine hydrochloride))-terminated surfaces were not cytotoxic.

The miR-17 family links p63 protein to MAPK signaling to promote the onset of human keratinocyte differentiation.

The p63 protein plays a key role in regulating human keratinocyte proliferation and differentiation. Although some p63-regulating microRNAs (miRNAs) have been identified in the control of epidermal homeostasis, little is known about miRNAs acting downstream of p63. In this paper, we characterized multiple p63-regulated miRNAs (miR-17, miR-20b, miR-30a, miR-106a, miR-143 and miR-455-3p) and elucidated their roles in the onset of keratinocyte differentiation.

Delta-like 4 inhibits choroidal neovascularization despite opposing effects on vascular endothelium and macrophages.

Inflammatory neovascularization, such as choroidal neovascularization (CNV), occur in the presence of Notch expressing macrophages. DLL4s anti-angiogenic effect on endothelial cells (EC) has been widely recognized, but its influence on Notch signaling on macrophages and its overall effect in inflammatory neovascularization is not well understood. We identified macrophages and ECs as the main Notch 1 and Notch 4 expressing cells in CNV.

Cardiac glycosides exert anticancer effects by inducing immunogenic cell death.

Some successful chemotherapeutics, notably anthracyclines and oxaliplatin, induce a type of cell stress and death that is immunogenic, hence converting the patient's dying cancer cells into a vaccine that stimulates antitumor immune responses. By means of a fluorescence microscopy platform that allows for the automated detection of the biochemical hallmarks of such a peculiar cell death modality, we identified cardiac glycosides (CGs) as exceptionally efficient inducers of immunogenic cell death, an effect that was associated with the inhibition of the plasma membrane Na(+)- and K(+)-dependent adenosine triphosphatase (Na(+)/K(+)-ATPase). CGs exacerbated the antineoplastic effects of DNA-damaging agents in immunocompetent but not immunodeficient mice.

Characterization of the transcriptional signature of C/EBPbeta isoforms (LAP/LIP) in Hep3B cells: implication of LIP in pro-survival functions.

Background & Aims: C/EBPbeta is an important mediator of several cellular processes, such as differentiation, proliferation, and survival of hepatic cells. However, a complete catalog of the targets of C/EBPbeta or the mechanism by which this transcription factor regulates certain liver-dependent pathways has not been clearly determined. Two major natural isoforms of this transcription factor exist: the liver-enriched activating protein (LAP) and the liver-enriched inhibitory protein (LIP), a functional LAP antagonist.

Small interfering RNAs induce target-independent inhibition of tumor growth and vasculature remodeling in a mouse model of hepatocellular carcinoma.

RNA interference mediated by small interfering RNAs (siRNAs) has emerged as a potential therapeutic approach to treat various diseases, including cancer. Recent studies with several animal models of posttraumatic revascularization demonstrated that synthetic siRNAs may produce therapeutic effects in a target-independent manner through the stimulation of the toll-like receptor-3 (TLR3)/interferon pathway and suppression of angiogenesis. To analyze the impact of siRNAs on tumor angiogenesis, we injected transgenic mice developing hepatocellular carcinoma (HCC) with either control siRNAs or siRNA targeting neuropilin-1.

Platelet JNK1 is involved in secretion and thrombus formation.

The role of c-Jun NH(2)-terminal kinase 1 (JNK1) in hemostasis and thrombosis remains unclear. We show here, with JNK1-deficient (JNK1(-/-)) mice, that JNK1 plays an important role in platelet biology and thrombus formation. In tail-bleeding assays, JNK1(-/-) mice exhibited longer bleeding times than wild-type mice (396 +/- 39 seconds vs 245 +/- 32 seconds).

Cross-talk between the VEGF-A and HGF signalling pathways in endothelial cells.

Background Information: Endothelial cells play a major role in angiogenesis, the process by which new blood vessels arise from a pre-existing vascular bed. VEGF-A (vascular endothelial growth factor-A) is a key regulator of angiogenesis during both development and in adults. HGF (hepatocyte growth factor) is a pleiotropic cytokine that may promote VEGF-A-driven angiogenesis, although the signalling mechanisms underlying this co-operation are not completely understood.

Neuropilin-1 and neuropilin-2 act as coreceptors, potentiating proangiogenic activity.

Neuropilin-1 and -2 (NRP1 and NRP2) are the transmembrane glycoproteins interacting with 2 types of ligands: class III semaphorins and several members of the VEGF family, the main regulators of blood and lymphatic vessel growth. We show here that both NRP1 and NRP2 can also bind hepatocyte growth factor (HGF). HGF is a pleiotropic cytokine and potent proangiogenic molecule that acts on its target cells by binding to the c-met receptor.

PSGL-1-mediated activation of EphB4 increases the proangiogenic potential of endothelial progenitor cells.

Endothelial progenitor cell (EPC) transplantation has beneficial effects for therapeutic neovascularization; however, only a small proportion of injected cells home to the lesion and incorporate into the neocapillaries. Consequently, this type of cell therapy requires substantial improvement to be of clinical value. Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors and their ephrin ligands are key regulators of vascular development.