The inner junction protein CFAP20 functions in motile and non-motile cilia and is critical for vision

Motile and non-motile cilia are associated with mutually-exclusive genetic disorders. Motile cilia propel sperm or extracellular fluids, and their dysfunction causes primary ciliary dyskinesia. Non-motile cilia serve as sensory/signalling antennae on most cell types, and their disruption causes single-organ ciliopathies such as retinopathies or multi-system syndromes. CFAP20 is a ciliopathy candidate known to modulate motile cilia in unicellular eukaryotes. We demonstrate that in zebrafish, cfap20 is required for motile cilia function, and in C. elegans, CFAP-20 maintains the structural integrity of non-motile cilia inner junctions, influencing sensory-dependent signalling and development. Human patients and zebrafish with CFAP20 mutations both exhibit retinal dystrophy. Hence, CFAP20 functions within a structural/functional hub centered on the inner junction that is shared between motile and non-motile cilia, and is distinct from other ciliopathy-associated domains or macromolecular complexes. Our findings suggest an uncharacterised pathomechanism for retinal dystrophy, and potentially for motile and non-motile ciliopathies in general.</p

Efficacy and Safety of Artificial Tears Containing Lipidure and Hypromellose for the Treatment of Moderate Dry Eye Disease in Contact Lens Wearers

Background and Objectives: Dry eye disease (DED) affects 5–50% of the global population and deeply influences everyday life activities. This study compared the efficacy, tolerability, and safety of novel Respilac artificial tears containing lipidure and hypromellose (HPMC) with the widely used Nextal artificial tears, which are also HPMC-based, for the treatment of moderate DED in contact lenses (CL) wearers. Materials and Methods: In a prospective, single-center, randomized investigation, 30 patients aged ≥18 years, diagnosed with moderate DED, and wearing CL were randomly assigned to the Respilac (n = 15) or Nextal group (n = 15). Patients self-administrated one drop of Respilac or Nextal in both eyes three times daily for 21 days. Changes in the endpoint (visual analogue scale (VAS) score for ocular tolerability, symptom assessment in dry eye (SANDE) score, non-invasive first break-up time (NIF-BUT) results, tear analysis value, meibography results, and CL tolerability results were assessed, comparing treatment groups and time-point evaluations. Adverse events (AEs) were also recorded and evaluated. Results: VAS scores decreased with time (p p = 0.13). Improvements were also detected from screening to end-of-treatment, which were indicated by the SANDE scores for severity and frequency (p p Conclusions: Respilac was confirmed to be effective, safe, and well-tolerated. Lipidure-based ophthalmic solution was shown not to be inferior to the currently used Nextal, however, showing improvements in DED symptoms. Within the existing literature, our study is one of the first to report that MPC plus HPMC-containing eye drops are an effective option for the treatment of moderate dry eye disease and desiccation damage prevention in contact lens wearers

Pro-inflammatory effect in 3T3-L1 mature adipocytes.

<p>In mature adipocytes, mRNA levels of Leptin <b>(A)</b>, IL6 <b>(B)</b>, IFNγ <b>(C)</b>, TNFα <b>(D)</b> and adiponectin <b>(E)</b> were assayed at day 8, the end of adipogenesis, by Real-time RT-PCR analysis, and expressed as Relative Expression Unit (REU). Bars represent the mean ± SD of four independent experiments. Asterisk indicates statistically significant difference (*p<0.05) between adipocytes cultured upon BPA treatment compared to controls.</p

Effect of BPA on 3T3-L1 proliferation and mRNA gene expression.

<p><b>(A)</b> 3T3-L1 pre-adipocyte were counted and expressed as cells/ml, at days 15, 16, 17 and 18, after 2 weeks of incubation with (BPA) or without (CTR) BPA 1nM, before adipogenesis started. PPARγ <b>(B)</b>, FABP4/AP2 <b>(C)</b> and cEBPα <b>(D)</b> mRNA levels were assayed during adipogenesis at days 0, 4 and 8, by Real-time RT-PCR analysis, expressed as Relative Expression Unit (REU). Bars represent the mean ± SD of four independent experiments. Asterisks indicate statistically significant differences (*p<0.05; **p<0.01; ***p<0.001) at days 4 and 8 compared to untreated day 0 for PPARγ <b>(B)</b>, at day 8 compared to untreated day 4 for FABP4/AP2 <b>(C)</b>, and at day 0 and day 4 compared to untreated day 0 for cEBPα <b>(D)</b>, without or with BPA incubation. Hashes (# p< 0.05; ### p<0.001) express statistically significant differences between day 8 with or without BPA incubation <b>(B and C)</b> and between day 0 and day 4 with or without BPA incubation <b>(D)</b>.</p

BPA effect on cellular neutral lipid droplet accumulation and glucose utilization in 3T3-L1 mature adipocytes.

<p>The microphotographs were obtained with an optical microscope in two original magnifications (10X and 20X), following ORO staining <b>(A)</b> in adipocytes upon BPA incubation compared to control cells. Lipid quantification test <b>(B)</b> was expressed as optical density (OD). Insulin stimulated glucose utilization test, expressed as fold over basal, was shown in differentiated 3T3-L1 cells incubated with 1 nM of BPA <b>(C)</b>. Bars represent the mean ± SD of four independent experiments. Asterisks indicate statistically significant differences (*p<0.05 and ***p<0.001) between adipocytes cultured upon BPA compared to controls.</p

BPA effect on insulin transduction pathway.

<p>Insulin signaling was tested in 3T3-L1 pre-adipocytes, before differentiation started (day 0; <b>A-C</b>) and in mature adipocytes (day 8; <b>B-D</b>). ERK1/2 (<b>A-B</b>) and PKB/AKT (<b>C-D</b>) phosphorylation after insulin stimulation were shown in untreated and BPA treated cells. Bars represent the mean ± SD of four independent experiments and blot is representative of four different experiments. Asterisks indicate statistically significant difference (*p<0.05, **p<0.01 and ***p<0.001) in adipocytes cultured upon BPA compared to controls.</p

Effect of BPA on PBMCs proliferation.

<p>Freshly isolated PBMCs (n = 12) were incubated in absence or in presence of BPA at the indicated concentrations (0.1 and 1nM). After 48 hours of incubation, cells were pulsed with [3-H]-thymidine for 16 hours before the harvesting. Results were expressed in cpm (count per minute). Experiments were performed on (A) unstimulated PBMCs or (B) under stimulation with phytohemagglutinin (PHA; 0.5μg/mL), or (C) with immobilized anti-CD3 (10μg/mL) and soluble anti-CD28 (1μg/ml) antibodies. Each dot represents mean value of triplicate wells. Bars indicate median values (n = 12). Asterisks indicate statistically significant differences (*p<0.05 and **p<0.01) by non-parametric Friedman test, followed by the Wilcoxon test for dependent samples.</p