Plasma Membrane Calcium ATPase-Neuroplastin Complexes Are Selectively Stabilized in GM1-Containing Lipid Rafts

The recent identification of plasma membrane (Ca2+)-ATPase (PMCA)-Neuroplastin (Np) complexes has renewed attention on cell regulation of cytosolic calcium extrusion, which is of particular relevance in neurons. Here, we tested the hypothesis that PMCA-Neuroplastin complexes exist in specific ganglioside-containing rafts, which could affect calcium homeostasis. We analyzed the abundance of all four PMCA paralogs (PMCA1-4) and Neuroplastin isoforms (Np65 and Np55) in lipid rafts and bulk membrane fractions from GM2/GD2 synthase-deficient mouse brains. In these fractions, we found altered distribution of Np65/Np55 and selected PMCA isoforms, namely PMCA1 and 2. Cell surface staining and confocal microscopy identified GM1 as the main complex ganglioside co-localizing with Neuroplastin in cultured hippocampal neurons. Furthermore, blocking GM1 with a specific antibody resulted in delayed calcium restoration of electrically evoked calcium transients in the soma of hippocampal neurons. The content and composition of all ganglioside species were unchanged in Neuroplastin-deficient mouse brains. Therefore, we conclude that altered composition or disorganization of ganglioside-containing rafts results in changed regulation of calcium signals in neurons. We propose that GM1 could be a key sphingolipid for ensuring proper location of the PMCA-Neuroplastin complexes into rafts in order to participate in the regulation of neuronal calcium homeostasis.

Outcomes of Sjögren’s versus non-Sjögren’s related dry eye in a longitudinal, tertiary clinic-based sample

Purpose To assess the long-term treatment outcomes of dry eye in patients with and without underlying primary Sjögren’s syndrome (SS). Design Retrospective longitudinal cohort. Methods SS and non-SS dry eye patients with clinic visits for a minimum of 5 consecutive years at a tertiary, dedicated dry eye clinic were included. Electronic health records were reviewed to collect data regarding demographics, objective dry eye parameters, treatments utilized at baseline and final visit, and corneal complications observed during follow-up. Results Two hundred and two patients (101 SS and 101 randomly selected non-SS), with a mean follow-up of 7.1 years were included. At baseline, mean conjunctival lissamine green staining score was 2.9 and mean corneal fluorescein staining score was 2.0. At last visit, notable improvement in staining score for cornea (–1.1, P < .001) and conjunctiva (–1.8, P < .001) was seen equally in both dry eye groups. Most patients (88.1%) had an escalation of treatment by the final visit, with similar rates in both groups (P = .51). Half (48.9%) of the patients had no conjunctival staining, and a third (34.4%) had no corneal staining at their last visit. Twenty (9.9%) patients experienced a vision-threatening corneal complication, including ulcers and melt, with no difference in occurrences between the groups (P = .64). Conclusions The majority of patients in this longitudinal, tertiary clinic-based sample demonstrated improvement in their ocular surface staining score by the final visit with escalation in treatment. Treatments used, improvement achieved, and corneal complication rates leading to loss of vision were similar in both SS and non-SS dry eye groups.

Efficacy of Intense Pulsed Light Treatment in Patients with Sjögren’s Syndrome Associated with Meibomian Gland Dysfunction

Purpose: To investigate the efficacy of intense pulsed light (IPL) treatment in patients with meibomian gland dysfunction (MGD) associated with Sjögren’s syndrome.Methods: This study included 43 patients with MGD and Sjögren’s syndrome. Patients received either IPL with meibomian gland expression (IPL/MGX) (n = 22) or MGX only (n = 21). Treatments were administered three times at a 3-week interval. Patients were followed up 6 weeks after the end of the treatment. Ocular Surface Disease Index (OSDI), tear film breakup time with fluorescein dye (FBUT), non-invasive tear breakup time, tear meniscus height (TMH), Schirmer test, SICCA ocular surface staining score, meibum quality score (MQS), and meibum expression score (MES) were evaluated at each visit. Meibomian gland dropouts (meiboscore) and tear film lipid layer grade were measured using keratography.Results: OSDI, FBUT, corneal surface staining score, MQS, meiboscore, and tear film lipid layer grade improved after IPL/MGX treatment (p < 0.05). In both treatment groups, MES significantly improved (p < 0.01 and p < 0.05 for IPL/MGX and MGX groups, respectively). The Schirmer test score, conjunctival surface staining score, and TMH after treatment were not significantly different between the groups. After treatment, the IPL/MGX group had significantly lower OSDI, FBUT, corneal staining score, MQS, and MES, but higher FBUT, compared with the MGX group (p < 0.05).Conclusions: IPL treatment effectively improved tear film, ocular surface parameters, meibomian gland function, and lipid layer grade in patients with Sjögren’s syndrome and MGD.

Three-year Randomized Prospective Clinical Trial of Class II Restorations Using Flowable Bulk-fill Resin Composites

SUMMARY Objectives: This randomized, prospective, and split-mouth study aimed to evaluate flowable bulk-fill resin composites in class II restorations and to compare with a conventional layering technique after a 3-year follow-up. Methods and Materials: Fifty-three subjects received three class II restorations according to the restorative systems: conventional microhybrid resin composite (PA, Peak Universal + Amelogen Plus, Ultradent), flowable bulk-fill and nanoparticulate resin composites (ABF, Adper Single Bond 2 + Filtek Bulk Fill Flow + Filtek Z350XT, 3M Oral Care), and flowable bulk-fill and microhybrid resin composites (XST, XP Bond + SDR + TPH3, Dentsply). The clinical performance and interproximal contacts were evaluated. Statistical analyses were performed using the equality test of two proportions, Logistic regression analysis, Friedman, Wilcoxon, Kruskal-Wallis, and Mann-Whitney tests (α=0.05). Results: Forty-seven patients were evaluated at 3 years. XST bulk-fill restorative system presented higher marginal discoloration than PA, and the opposite occurred for surface staining. All restorative systems resulted in decreased interproximal contacts, occurring early for XST. Conclusions: Although the restorative system using incremental technique presented better performance for marginal discoloration, one of the restorative systems that used flowable bulk-fill resin composite (XST) showed the lowest surface staining. All restorative systems had decreased proximal contact over time.

A Two-Year Comparative Evaluation of Clinical Performance of a Nanohybrid Composite Resin to a Flowable Composite Resin

Objective: This prospective in vivo study aimed to compare the clinical behavior of a flowable composite resin (Genial Universal Flo, GC) and a nanohybrid universal composite resin (Tetric Evo Ceram, Ivoclar Vivadent) used in Class I and II direct esthetic restorations in posterior teeth. Methods: A total of 108 Class I and II direct restorations were performed in patients aged between 20 and 60 years. The originality of this study lies in the fact that both materials were placed in pairs, in the same clinical environment (i.e., the same patient and the same type of tooth). The evaluations were performed now of restoration and after 2-weeks, 6-, 12-, and 24-months intervals using clinical examination, clinical photographs, and radiological examination, according to modified USPHS criteria. Statistical analysis was performed using the Fisher exact test and chi-square analysis. Results: At baseline, the universal composite resin showed better esthetic properties such as surface luster, surface staining marginal staining. Both materials regressed significantly over time with no significant difference between groups. Conclusions: Both flowable and nanohybrid composite resins exhibit acceptable clinical performance. The present 24 months of evaluation of different composites showed that the G-ænial Universal Flo could be an effective esthetic material for posterior restoration. No significant difference between both materials over time concerning surface luster, surface staining, and marginal staining.

Fit-for-purpose based testing and validation of antibodies to amino- and carboxy-terminal domains of cannabinoid receptor 1

Specific and selective anti-CB1 antibodies are among the most powerful research tools to unravel the complex biological processes mediated by the CB1 receptor in both physiological and pathological conditions. However, low performance of antibodies remains a major source of inconsistency between results from different laboratories. Using a variety of techniques, including some of the most commonly accepted ones for antibody specificity testing, we identified three of five commercial antibodies against different regions of CB1 receptor as the best choice for specific end-use purposes. Specifically, an antibody against a long fragment of the extracellular amino tail of CB1 receptor (but not one against a short sequence of the extreme amino-terminus) detected strong surface staining when applied to live cells, whereas two different antibodies against an identical fragment of the extreme carboxy-terminus of CB1 receptor (but not one against an upstream peptide) showed acceptable performance on all platforms, although they behaved differently in immunohistochemical assays depending on the tissue fixation procedure used and showed different specificity in Western blot assays, which made each of them particularly suitable for one of those techniques. Our results provide a framework to interpret past and future results derived from the use of different anti-CB1 antibodies in the context of current knowledge about the CB1 receptor at the molecular level, and highlight the need for an adequate validation for specific purposes, not only before antibodies are placed on the market, but also before the decision to discontinue them is made.

A Comparison of Symptoms and Signs in Patients with Inflammatory Dry Eye Disease using two Commercially Available Treatment Formulations of Cyclosporine A

Purpose: Dry eye (DED) is a multifactorial disease of the ocular surface in which ocular surface inflammation and damage play etiological roles. In the present study, we compared symptoms and signs in patients with DED treated with 0.1% Cyclosporine A (CsA) cationic emulsion (Ikervis, Santen) and 0.05% CsA anionic emulsion (Restasis, Allergan).Methods: Single-center, retrospective, 202 consecutive DED patients were treated with 0.1% CsA cationic emulsion (Ikervis, n=101) or 0.05% CsA anionic emulsion (Restasis, n=101) over six months. Ophthalmological work-up included Ocular Surface Disease Index (OSDI) questionnaire, fluorescein break-up time (FBUT), fluorescein ocular surface staining (OSS), Schirmer’s test, meibum expressibility (ME), and meibum quality (MQ). Results: In both treatment groups, subgroup analysis revealed a significant improvement of OSDI only in patients with severe symptoms at baseline (OSDI 33-100). Ikervis-treated patients with OSDI <23 at baseline had a significant increase in symptoms. OSS and FBUT improved significantly in most subgroups irrespective of Ikervis or Restasis being used. Regression analysis revealed a significant superiority to Restasis with regard to FBUT improvement and superiority to Ikervis in regards ME improvement. Conclusions: We conclude that there is seemingly no substantial difference in efficacy between the two drugs. The strength of Restasis is increased efficacy at improving FBUT and of Ikervis improving ME.

Ocular benzalkonium chloride exposure: problems and solutions

Preservatives in multidose formulations of topical ophthalmic medications are crucial for maintaining sterility but can be toxic to the ocular surface. Benzalkonium chloride (BAK)—used in approximately 70% of ophthalmic formulations—is well known to cause cytotoxic damage to conjunctival and corneal epithelial cells, resulting in signs and symptoms of ocular surface disease (OSD) including ocular surface staining, increased tear break-up time, and higher OSD symptom scores. These adverse effects are more problematic with chronic exposure, as in lifetime therapy for glaucoma, but can also manifest after exposure as brief as seven days. Multiple strategies are available to minimize or eliminate BAK exposure, among them alternative preservatives, preservative-free formulations including sustained release drug delivery platforms, and non-pharmacological therapies for common eye diseases and conditions. In this paper, we review the cytotoxic and clinical effects of BAK on the ocular surface and discuss existing and emerging options for ocular disease management that can minimize or eliminate BAK exposure.

Extended live-cell barcoding approach for multiplexed mass cytometry

Sample barcoding is essential in mass cytometry analysis, since it can eliminate potential procedural variations, enhance throughput, and allow simultaneous sample processing and acquisition. Sample pooling after prior surface staining termed live-cell barcoding is more desirable than intracellular barcoding, where samples are pooled after fixation and permeabilization, since it does not depend on fixation-sensitive antigenic epitopes. In live-cell barcoding, the general approach uses two tags per sample out of a pool of antibodies paired with five palladium (Pd) isotopes in order to preserve appreciable signal-to-noise ratios and achieve higher yields after sample deconvolution. The number of samples that can be pooled in an experiment using live-cell barcoding is limited, due to weak signal intensities associated with Pd isotopes and the relatively low number of available tags. Here, we describe a novel barcoding technique utilizing 10 different tags, seven cadmium (Cd) tags and three Pd tags, with superior signal intensities that do not impinge on lanthanide detection, which enables enhanced pooling of samples with multiple experimental conditions and markedly enhances sample throughput.