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Background: Gene expression profiling (GEP)-based prognostic signatures are being rapidly integrated into clinical decision making for systemic management of breast cancer patients. However, GEP remains relatively underdeveloped for locoregional risk assessment. Yet, locoregional recurrence (LRR), especially early after surgery, is associated with poor survival., Patients and Methods: GEP was carried out on two independent luminal-like breast cancer cohorts of patients developing early (≤5 years after surgery) or late (>5 years) LRR and used, by a training and testing approach, to build a gene signature able to intercept women at risk of developing early LRR. The GEP data of two in silico datasets and of a third independent cohort were used to explore its prognostic value., Results: Analysis of the first two cohorts led to the identification of three genes, CSTB, CCDC91 and ITGB1, whose expression, derived by principal component analysis, generated a three-gene signature significantly associated with early LRR in both cohorts (P value <0.001 and 0.005, respectively), overcoming the discriminatory capability of age, hormone receptor status and therapy. Remarkably, the integration of the signature with these clinical variables led to an area under the curve of 0.878 [95% confidence interval (CI) 0.810-0.945]. In in silico datasets we found that the three-gene signature retained its association, showing higher values in the early relapsed patients. Moreover, in the third additional cohort, the signature significantly associated with relapse-free survival (hazard ratio 1.56, 95% CI 1.04-2.35)., Conclusions: Our three-gene signature represents a new exploitable tool to aid treatment choice in patients with luminal-like breast cancer at risk of developing early recurrence., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Purpose: The aim was to develop a method to assess the electrostatic properties of human tear samples, and to evaluate their modifications induced by the wear of contact lenses (CLs)., Method: The barrier method was developed for the measurement of the isoelectric point (IEP) on relatively small quantities. The method was applied to compare three groups: tears (T NW ) of non-wearers, tears (T W&#95;etaf ) of regular wearers of etafilcon A CLs, and tears (T W&#95;omaf ) of regular wearers of omafilcon A CLs. Zeta potential (ζ) as a function of pH was measured by a Zetasizer Nano ZS90 (Malvern Instruments) on 40%-diluted samples, obtained by mixing 57 μL of tears of different subjects of the same group with 85 μL of HCl aqueous solution. IEP was deduced as the pH at which ζ is zero, i.e. the net electric charge on tear constituents being neutralized., Results: Within an error of about 0.05, IEPs were found to be 2.90 (T NW ), 2.80 (T W&#95;omaf ), and 3.16 (T W&#95;etaf ). On average, a lower H + concentration is needed to neutralize the surface charge of the tear components of etafilcon A wearers, compared to both T NW and T W&#95;omaf ., Conclusion: IEP measurements on tear samples of wearers of different types of CLs are proposed in order to enhance the knowledge on the modifications of the profile of charged species in tears. The T W&#95;etaf results, compared to those of the other groups, are compatible with an increase, due to the wear of etafilcon A CLs, of the relative concentration of high-IEP proteins., (Copyright © 2019. Published by Elsevier Ltd.)

Lactoferrin (LF), an iron-binding protein with antioxidant activity, is significantly reduced in the lacrimal film of patients affected by keratoconus (KC) compared to healthy subjects, and this is supposed to be the cause for tear iron increase and consequent iron deposition and oxygen free radical accumulation in the cornea. We decided to study if LF-loaded contact lenses (LF-CLs) could exert antioxidant activity on epithelial cells incubated in tears collected from two patients affected by keratoconus (KC1 and KC2). Moreover through this model we indirectly estimated iron concentration in the tears of healthy and KC subjects. Reflex tears were collected during the first 2 min of light or onion-induced lacrimation and stored at -20 °C. After incubation in tears for 18 h, mortality of epithelial cells was investigated by trypan blue exclusion test. Successively, LF-CLs were deposed on cells incubated in KC1 tears. For the indirect determination of iron content, cells were incubated with LF 1.2 mg/mL and different FeSO 4 concentrations, and for the estimation of iron from the patient's tears, cells were incubated with free serum medium and healthy tears (1:1) and different FeSO 4 concentrations. Epithelial cells incubated with reflex tears of KC patients showed increased mortality (27.7 ± 3.9%, p = 0.0003, for KC1 and 17.6 ± 0.95%, p = 0.014, for KC2) compared to epithelial cells maintained in control healthy tears (8.6 ± 1.2%). This difference in mortality was correlated with tear iron concentration, which was estimated at 4.58 μg/mL for the healthy subjects, at 56.28 μg/mL for KC1, and at 8.7 μg/mL for KC2 patient. Application of LF-CLs counteracted KC tear cytotoxicity restoring viability obtained in the presence of control tears. Therapeutic contact lenses obtained by LF loading can reduce oxidative stress induced by patients' tears and might represent an efficient device to arrest the progression of keratoconus., (Copyright © 2018 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

Purpose: The aim is discussing the origins of worsening of external eye condition (EEC) and of tear film (TF) instability after wear of silicone-hydrogel contact lenses (CLs) with hydrogen-peroxide (H 2 O 2 ) care system., Methods: EEC and TF stability were evaluated before and after 15days of wear combined with different care systems: (1) H 2 O 2 , (2) detergent solution and H 2 O 2 , (3) multipurpose solution (MPS), (4) H 2 O 2 and artificial tears. In-vitro cell mortality tests were performed after 24h cell incubation with CLs treated with H 2 O 2 . Photon correlation spectroscopy (PCS) was carried out on tears of non-wearers and CL wearers who used MPS or H 2 O 2 solution., Results: Worsening of EEC was observed only for the group using H 2 O 2 (group 1). In-vitro, cell mortality was found higher for worn CL than for unworn CLs. Worsening of TF stability was observed regardless of care system and also PCS results on tears of CL wearers were found different compared to non-wearers regardless of care system. The only observed remedy for tear instability of CL wearers was found to be the administration of artificial tears., Conclusions: Worsening of EEC of CL wearers using H 2 O 2 is attributed to H 2 O 2 scarce cleaning efficacy, which can be solved by adding a CL detergent solution. The origin of TF instability is found to be different. A remedy was found to be the administration of artificial tears, whose effect could be attributed either to the role of specific components or to rinsing and replacement of TF during wear., (Copyright © 2017 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.)

This study aims to deepen the knowledge on tear film properties by the development of a protocol for analyses of Photon Correlation Spectroscopy (PCS) on human tears and by the comparison between PCS results obtained on tears of contact lens wearers and non-wearers. Tears (5μL) were collected by a glass capillary. The analyses provide the hydrodynamic diameter of tear components by analyzing intensity fluctuations in time of scattered light. PCS appears a promising technique for studying tear features and for shedding light on specific eye conditions, such as on the clinical effects of CL wear. In fact, statistical difference (p<0.001) was found between the measured mean hydrodynamic diameter of tear components of wearers and non-wearers, the resulting value significantly higher for CL wearers. The scenario does not substantially change after (25±5)min from the CL removal. The difference is attributed to changes in the interactions between tear constituents due to CL wear. In order to get deeper insights on the influence of CL wear on aggregation and structure of tear components, a preliminary Electron Spin Resonance (ESR) investigation was performed, monitoring Fe 3+ species. ESR spectra on tears of both CL wearers and non-wearers showed the presence of intense signals, probably associated to iron (III) centers in proteins such as lactoferrin, and a weaker resonance attributable to Fe 3+ species interacting with S-S bridges of lysozyme. Differences in ESR spectra between CL wearers and non-wearers were detected and tentatively ascribed to changes in coordination or in local environment of Fe 3+ centers connected to aggregation phenomena induced by CL wear, which promote their interaction with other neighboring iron species., (Copyright © 2017. Published by Elsevier B.V.)

Myotonia congenita is an autosomal dominantly or recessively inherited muscle disorder causing impaired muscle relaxation and variable degrees of permanent muscle weakness, abnormal currents linked to the chloride channel gene (CLCN1) encoding the chloride channel on skeletal muscle membrane. We describe 12 novel mutations: c.1606G>C (p.Val536Leu), c.2533G>A (p.Gly845Ser), c.2434C>T (p.Gln812X), c.1499T>G (p.E500X), c.1012C>T (p.Arg338X), c.2403+1G>A, c.2840T>A (p.Val947Glu), c.1598C>T (p.Thr533Ile), c.1110delC, c.590T>A (p.Ile197Arg), c.2276insA Fs800X, c.490T>C (p.Trp164Arg) in 22 unrelated Italian patients. To further understand the functional outcome of selected missense mutations (p.Trp164Arg, p.Ile197Arg and p.Gly845Ser, and the previously reported p.Gly190Ser) we characterized the biophysical properties of mutant ion channels in tsA cell model. In the physiological range of muscle membrane potential, all the tested mutations, except p.Gly845Ser, reduced the open probability, increased the fast and slow components of deactivation and affected pore properties. This suggests a decrease in macroscopic chloride currents impairing membrane potential repolarization and causing hyperexcitability in muscle membranes. Detailed clinical features are given of the 8 patients characterized by cell electrophysiology. These data expand the spectrum of CLCN1 mutations and may contribute to genotype-phenotype correlations. Furthermore, we provide insights into the fine protein structure of ClC-1 and its physiological role in the maintenance of membrane resting potential., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Neuronal nicotinic acetylcholine receptors (nAChRs) are Ca(2+)-permeable ligand-gated channels widely expressed in the central and peripheral nervous system. One of the most Ca(2+) selective isoform is the homopentameric alpha7-nAChR implicated in schizophrenia. The activity of alpha7-nAChRs is usually recorded electrophysiologically, which limits the amount of information obtained. Here, we used fluorescence imaging to record Ca(2+) transients associated with activation of the alpha7-nAChR in neuroblastoma cells stably expressing human alpha7-nAChRs. Application of nicotine (50 microM) consistently evoked transient (30s), stereotyped Ca(2+) responses that were inhibited by the selective alpha7-nAChRs antagonists methyllycaconitine (MLA) and alpha-bungarotoxin, and greatly increased and prolonged by the allosteric modulator PNU-120596 (1 microM). Unexpectedly, brief (1-5s), repetitive Ca(2+) transients of sub-micrometric dimension were observed in filopodia of cells expressing alpha7-nAChR. PNU-120596 increased the frequency and slowed the decay kinetics of these miniature Ca(2+) elevations, which were insensitive to ryanodine, preserved during hyperpolarisation, and prevented by MLA, alpha-bungarotoxin, or Ca(2+) removal. Global Ca(2+) responses were also recorded in ganglion cells of embryo chicken retina during co-application of PNU-120596 and nicotine, together with whole-cell currents and brief current bursts. These data demonstrate that Ca(2+) signals generated by alpha7-nAChRs can be recorded optically both in cell lines and in intact tissues. The possibility to image miniature Ca(2+) signals enables to map the location of functional alpha7-nAChR channel clusters within cells and to analyze their single channel properties optically. Deciphering the rich pattern of intracellular Ca(2+) signals generated by the activity of the alpha7-nAChRs will reveal the physiological role of these receptor-channels.