Preparation of human primary macrophages to study the polarization from monocyte-derived macrophages to pro- or anti-inflammatory macrophages at biomaterial interface in vitro

Background/purpose: Testing of dental materials when in contact with innate immune cells has been so far hindered by the lack of proper in vitro models. Human primary monocyte-derived macrophages (MDMs) would be an excellent option to this aim. However, the inability to detach them from the tissue culture plates contrast the possibility to culture them on biomaterials. The goal of the present work is to present and validate an innovative protocol to obtain MDMs from peripheral blood monocytes, and to reseed them in contact with biomaterials without altering their viability and phenotype. Materials and methods: We differentiated MDMs on ultra-low attachment tissue culture plastics and recovered them with specific detachment solution in order to be reseeded on a secondary substrate. Therefore, using biological assays (RT-PCR, Western blot, and immunofluorescence) we compared their phenotype to MDMs differentiated on standard culture plates. Results: Transferred MDMs keep their differentiated M0 resting state, as well as the ability to be polarized into M1 (pro-inflammatory) or M2 (anti-inflammatory) macrophages. Conclusion: These data provide the dental material research community the unprecedented possibility to investigate the immunomodulatory properties of biomaterials for dental application

Expression of ras oncogene p21 protein in normal and neoplastic laryngeal tissues: correlation with histopathological features and epidermal growth factor receptors.

Western blotting analysis of the p21 ras oncoprotein was performed in seven normal laryngeal mucosa specimens and 43 primary laryngeal cancers. Varying p21 levels, expressed as optical density (OD), were found in normal mucosa (median 1.94 OD, range 0.90-2.17 OD) and in primary laryngeal tumours (median 1.74 OD, range 0.30-6.37 OD). When p21 expression in laryngeal cancer was compared with the normal counterpart, higher levels were found in neoplastic than in normal laryngeal tissue (median 2.54 OD, range 1.76-6.37 OD, vs median 1.94 OD, range 0.90-2.17 OD) (P = 0.023). Immunohistochemical analysis demonstrated that most of the tumour cells (more than 70%) were immunostained while the stromal component was unreactive. No correlation between p21 expression and tumour location, stage and histopathological grade was observed. The correlation between ras p21 protein expression and epidermal growth factor receptor (EGFR) levels was also investigated. EGFR-positive cases did not show any difference in p21 expression with respect to EGFR-negative cases (median 1.52 OD, range 0.30-6.37 OD, vs median 1.84 OD, range 0.93-3.71 OD). Our findings suggest that overexpression of p21 protein is associated with a malignant phenotype in laryngeal cancer. Further studies should be undertaken to evaluate whether the assessment of p21 protein expression may have clinical significance in laryngeal cancer

Nanoemulsions of Satureja montana essential oil. Antimicrobial and antibiofilm activity against avian Escherichia coli strains

Satureja montana essential oil (SEO) presents a wide range of biological activities due to its high content of active phytochemicals. In order to improve the essential oil’s (EO) properties, oil in water nanoemulsions (NEs) composed of SEO and Tween-80 were prepared, characterized, and their antimicrobial and antibiofilm properties assayed against Escherichia coli strains isolated from healthy chicken. Since surfactant and oil composition can strongly influence NE features and their application field, a ternary phase diagram was constructed and evaluated to select a suitable sur-factant/oil/water ratio. Minimal inhibitory concentration and minimal bactericidal concentration of NEs, evaluated by the microdilution method, showed that the SEO NE formulation exhibited higher inhibitory effects against planktonic E. coli than SEO alone. The quantification of biofilm production in the presence of NEs, assessed by crystal violet staining and scanning electron microscopy, evi-denced that sub-MIC concentrations of SEO NEs enable an efficient reduction of biofilm production by the strong producer strains. The optimized nanoemulsion formulation could ensure food safety quality, and counteract the antibiotic resistance of poultry associated E. coli, if applied/aerosolized in poultry farms

Effective In Vivo Topical Delivery of siRNA and Gene Silencing in Intact Corneal Epithelium Using a Modified Cell-Penetrating Peptide

Autosomal dominantly inherited genetic disorders such as corneal dystrophies are amenable to allele-specific gene silencing with small interfering RNA (siRNA). siRNA delivered to the cornea by injection, although effective, is not suitable for a frequent long-term treatment regimen, whereas topical delivery of siRNA to the cornea is hampered by the eye surface's protective mechanisms. Herein we describe an attractive and innovative alternative for topical application using cell-penetrating peptide derivatives capable of complexing siRNA non-covalently and delivering them into the cornea. Through a rational design approach, we modified derivatives of a cell-penetrating peptide, peptide for ocular delivery (POD), already proved to diffuse into the corneal layers. These POD derivatives were able to form siRNA-peptide complexes (polyplexes) of size and ζ-potential similar to those reported able to undergo cellular internalization. Successful cytoplasmic release and gene silencing in vitro was obtained when an endosomal disruptor, chloroquine, was added. A palmitoylated-POD, displaying the best delivery properties, was covalently functionalized with trifluoromethylquinoline, an analog of chloroquine. This modified POD, named trifluoromethylquinoline-palmitoyl-POD (QN-Palm-POD), when complexed with siRNA and topically applied to the eye in vivo, resulted in up to 30% knockdown of luciferase reporter gene expression in the corneal epithelium. The methods developed within represent a valid standardized approach that is ideal for screening of a range of delivery formulations

P2316Outcome of septal reduction therapies for obstructive hypertrophic cardiomyopathy in a high-flow referral centre with moderate volume procedural programmes

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CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting

CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann's epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics

Incidence of stroke in patients with hypertrophic cardiomyopathy in stable sinus rhythm during long-term monitoring.

Patients with hypertrophic cardiomyopathy (HCM) are at increased risk of stroke, but the incidence and factors associated with cardioembolic events in HCM patients without atrial fibrillation (AF) remain unresolved. We determined the incidence of stroke in patients in sinus rhythm (SR) monitored with a cardiac implantable electronic device (CIED). All consecutive patients diagnosed with HCM and referred to CIED implantation with >16 years at diagnosis and ≥ 1 year follow-up post CIED implantation were retrospectively reviewed. Severe LA dilatation was defined as ≥48 mm. Patients were stratified by rhythm as: Pre-existing AF (AF present prior to CIED); De novo AF (AF present after CIED implantation); SR: no episodes of AF. Of 1651 patients, 185 (11.2%) implanted with a CIED were included (57% men, age: 54 ± 17 years). Baseline, pre-existing AF was present in 73 (39%) patients. Ischemic stroke was reported in 19 (10.3%, 1.78%/year) patients and was similar across the three groups (2.3%/year vs 1.1%/year vs 0.6%/year in patients in SR vs pre-existing AF vs de novo AF, respectively, p = 0.235). In SR patients, a LAD≥48 mm posed the greatest risk of stroke (Hazard Ratio: 10.03,95% Confidence-Interval 2.79-16.01). At Cox multivariable analysis, after adjustment for oral anticoagulation, LA was independently associated with stroke while rhythm was not. in HCM patients with CIED long-term monitoring and no prior history of AF, stroke rates were similar in those with de novo AF or stable SR. Severe LA dilatation was a powerful risk factor, irrespective of AF

High Bone Mass Disorders : New Insights From Connecting the Clinic and the Bench

Monogenic high bone mass (HBM) disorders are characterized by an increased amount of bone in general, or at specific sites in the skeleton. Here, we describe 59 HBM disorders with 50 known disease-causing genes from the literature, and we provide an overview of the signaling pathways and mechanisms involved in the pathogenesis of these disorders. Based on this, we classify the known HBM genes into HBM (sub)groups according to uniform Gene Ontology (GO) terminology. This classification system may aid in hypothesis generation, for both wet lab experimental design and clinical genetic screening strategies. We discuss how functional genomics can shape discovery of novel HBM genes and/or mechanisms in the future, through implementation of omics assessments in existing and future model systems. Finally, we address strategies to improve gene identification in unsolved HBM cases and highlight the importance for cross-laboratory collaborations encompassing multidisciplinary efforts to transfer knowledge generated at the bench to the clinic. (c) 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).Peer reviewe