Light-Induced Thiol Oxidation of Recoverin Affects Rhodopsin Desensitization

The excessive light illumination of mammalian retina is known to induce oxidative stress and photoreceptor cell death linked to progression of age-related macular degeneration. The photochemical damage of photoreceptors is suggested to occur via two apoptotic pathways that involve either excessive rhodopsin activation or constitutive phototransduction, depending on the light intensity. Both pathways are dramatically activated in the absence of rhodopsin desensitization by GRK1. Previously, we have shown that moderate illumination (halogen lamp, 1,500 lx, 1–5 h) of mammalian eyes provokes disulfide dimerization of recoverin, a calcium-dependent regulator of GRK1. Here, we demonstrate under in vivo conditions that both moderate long-term (metal halide lamp, 2,500 lx, 14 h, rat model) and intense short-term (halogen lamp, 30,000 lx for 3 h, rabbit model) illumination of the mammalian retina are accompanied by accumulation of disulfide dimer of recoverin. Furthermore, in the second case we reveal alternatively oxidized derivatives of the protein, apparently including its monomer with sulfinic group. Histological data indicate that thiol oxidation of recoverin precedes apoptosis of photoreceptors. Both disulfide dimer and oxidized monomer (or oxidation mimicking C39D mutant) of recoverin exhibit lowered α-helical content and thermal stability of their apo-forms, as well as increased Ca2+ affinity. Meanwhile, the oxidized monomer and C39D mutant of recoverin demonstrate impaired ability to bind photoreceptor membranes and regulate GRK1, whereas disulfide dimer exhibits notably improved membrane binding and GRK1 inhibition in absence of Ca2+. The latter effect is expected to slow down rhodopsin desensitization in the light, thereby favoring support of the light-induced oxidative stress, ultimately leading to photoreceptor apoptosis. Overall, the intensity and duration of illumination of the retina affect thiol oxidation of recoverin likely contributing to propagation of the oxidative stress and photoreceptor damage

The problem of goal-setting in a foreign language teaching in a diverse educational environment

В статье поднимается проблема педагогического целеполагания при обучении иностранному языку в разнородной образовательной среде. Актуальность проблемы связана с процессами интернационализации образовательных систем, когда вопросы учета разнообразия обучающихся и проблема освоения ими «единого» глобального языка общения, в первую очередь английского, становятся одной из главных задач педагогической науки и практики. Авторы проводят анализ подходов к определению цели обучения, сравнивают различные трактовки понятия «целеполагание», определяют основные субъекты «педагогического целеполагания». В работе выделяются проблемы макро- и микроуровней постановки целей в обучении гетерогенных групп. Приводятся примеры формулирования целей курса и/или урока иностранного языка с позиций всех субъектов образовательного процесса. В статье даются рекомендации по соблюдению этапов целеполагания при реализации ФГОС школьного образования. Авторы делают вывод о необходимости постановки измеримых и достижимых целей, которые не только должны быть приняты обучающимися, но и исходить из их разнообразных образовательных потребностей с целью повышения эффективности процесса обучения иностранным . The article deals with the problem of pedagogical goal-setting in teaching foreign languages in a diverse educational environment. The importance of this problem is connected with the processes of internationalization of national educational systems when the issues of teaching diverse students arise and the importance of developing their foreign communicative competence by means of English as a ‘global’ language of communication becomes one of the main objectives of mainstream teaching and language teaching methodology. The authors analyse different approaches to determining educational aims and compare various definitions of a concept “goal-setting” as well as define main subjects of “pedagogical goal-setting”. This paper highlights the problems of stating educational objectives at macro- and micro- levels in teaching diverse groups of learners. The examples of formulating purposes of a language course or/and a lesson from the positions of all subjects of an educational process are given. The article provides some recommendations on how to follow the goal-setting stages while implementing national standards in education. The authors come to the conclusion that in order to increase the efficiency of foreign language teaching it is necessary to set measurable and achievable aims which have to be accepted by language learners and which should take into account different learners’ educational needs

Assessment of Bowel Preparation Using Low-Volume Sulphate-Based Preparations in Comparison with Macrogols: A Multicenter, Randomized, Comparative Clinical Study of the 3rd Phase

Oral sulphate solution (OSS: sodium sulphate, potassium sulphate and magnesium sulphate) is a low-volume osmotic agent for cleansing the intestines.Aim: in a multicentre, prospective, randomized, 3rd phase study with two parallel groups, the effectiveness, safety and tolerability of OSS was evaluated in comparison with Macrogol 4000 with electrolytes (a reference preparation for bowel cleansing in Russia) in adult patients who were scheduled for routine diagnostic colonoscopy.Methods. This study was conducted in three Russian research centres during the March–December, 2015 period. Men and women over the age of 18 scheduled to undergo routine diagnostic colonoscopy were randomly assigned either to the OSS group or to the Macrogol group with a fractional use mode before the colonoscopy. The colonoscopy researchers were not aware of which preparation had been taken by the patients. Anonymized video records were centrally analysed by three experts. The primary end point was the proportion of patients with a successful bowel preparation for colonoscopy ≥6 points, as determined by the Boston Bowel Preparation Scale of quality assessment (BBPS scale).Results. 296 patients were randomized in the study (147 patients were treated with OSS, 149 patients received Macrogol); 294 participants were included in the Intention to Treat population (ITT-population), and 274 participants were included in the population of patients who completed the study according to the protocol (Per-Protocol; PP-population) (139 patients received OSS, 135 patients received Macrogol). The proportion of patients with a successful bowel preparation (BBPS ≥6 scores) was high in both groups (OSS [PP-population]: 97.2 % (95 % confidence interval [CI] 89.5–99.3), Macrogol [PP-population]: 97.7 % (95 % CI: 90.7–99.4)). The corrected difference between the groups was -0.5 % (95 % CI: -4.2–3.3), thereby demonstrating “no less effective” of OSS as compared to Macrogol. Compliance with the drug use regime was higher in the OSS group than in the Macrogol group (95.7 % versus 82.3 %, respectively, p-value = 0.0011, ITT-population).The most common symptom reported in patients was nausea (27.9 % in the OSS group and 12.9 % in the Macrogol group). The proportion of patients who developed nausea was significantly higher in the OSS group than in the Macrogol group (25.2 % compared with 10.2 % when taking the first dose of the preparation (p = 0.0008) and 19.7 % compared with 6.8 % when taking the second dose of the preparation (p = 0.0016)). Differences in other symptoms (bloating, abdominal pain or abdominal discomfort) between the groups were not significant, with the severity of symptoms being generally mild. The safety profile of the investigated preparations in patients with inflammatory bowel disease (IBD) in remission did not differ from that in the general patient population.The differences in terms of secondary endpoints were not identified, including BBPS assessment for different sections of the colon, the level of polyp detection, the duration and completeness of colonoscopy, and the investigator’s satisfaction with the procedure. The analysis by subgroups also did not reveal any significant differences.Conclusion. In this study, the “not less effectiveness” of the sulphate solution was demonstrated as compared to Macrogol in a fractional use mode. Both preparations were well tolerated. Despite the higher incidence of nausea in the OSS group, the patients showed significantly higher compliance with the OSS mode as compared to that of Macrogol.This study is registered with the ClinicalTrials.gov Registry of Clinical Trials, No. NCT02321462

Assessment of Bowel Preparation Using Low-Volume Sulphate-Based Preparations in Comparison with Macrogols: A Multicenter, Randomized, Comparative Clinical Study of the 3rd Phase

Oral sulphate solution (OSS: sodium sulphate, potassium sulphate and magnesium sulphate) is a low-volume osmotic agent for cleansing the intestines.Aim: in a multicentre, prospective, randomized, 3rd phase study with two parallel groups, the effectiveness, safety and tolerability of OSS was evaluated in comparison with Macrogol 4000 with electrolytes (a reference preparation for bowel cleansing in Russia) in adult patients who were scheduled for routine diagnostic colonoscopy.Methods. This study was conducted in three Russian research centres during the March–December, 2015 period. Men and women over the age of 18 scheduled to undergo routine diagnostic colonoscopy were randomly assigned either to the OSS group or to the Macrogol group with a fractional use mode before the colonoscopy. The colonoscopy researchers were not aware of which preparation had been taken by the patients. Anonymized video records were centrally analysed by three experts. The primary end point was the proportion of patients with a successful bowel preparation for colonoscopy ≥6 points, as determined by the Boston Bowel Preparation Scale of quality assessment (BBPS scale).Results. 296 patients were randomized in the study (147 patients were treated with OSS, 149 patients received Macrogol); 294 participants were included in the Intention to Treat population (ITT-population), and 274 participants were included in the population of patients who completed the study according to the protocol (Per-Protocol; PP-population) (139 patients received OSS, 135 patients received Macrogol). The proportion of patients with a successful bowel preparation (BBPS ≥6 scores) was high in both groups (OSS [PP-population]: 97.2 % (95 % confidence interval [CI] 89.5–99.3), Macrogol [PP-population]: 97.7 % (95 % CI: 90.7–99.4)). The corrected difference between the groups was -0.5 % (95 % CI: -4.2–3.3), thereby demonstrating “no less effective” of OSS as compared to Macrogol. Compliance with the drug use regime was higher in the OSS group than in the Macrogol group (95.7 % versus 82.3 %, respectively, p-value = 0.0011, ITT-population).The most common symptom reported in patients was nausea (27.9 % in the OSS group and 12.9 % in the Macrogol group). The proportion of patients who developed nausea was significantly higher in the OSS group than in the Macrogol group (25.2 % compared with 10.2 % when taking the first dose of the preparation (p = 0.0008) and 19.7 % compared with 6.8 % when taking the second dose of the preparation (p = 0.0016)). Differences in other symptoms (bloating, abdominal pain or abdominal discomfort) between the groups were not significant, with the severity of symptoms being generally mild. The safety profile of the investigated preparations in patients with inflammatory bowel disease (IBD) in remission did not differ from that in the general patient population.The differences in terms of secondary endpoints were not identified, including BBPS assessment for different sections of the colon, the level of polyp detection, the duration and completeness of colonoscopy, and the investigator’s satisfaction with the procedure. The analysis by subgroups also did not reveal any significant differences.Conclusion. In this study, the “not less effectiveness” of the sulphate solution was demonstrated as compared to Macrogol in a fractional use mode. Both preparations were well tolerated. Despite the higher incidence of nausea in the OSS group, the patients showed significantly higher compliance with the OSS mode as compared to that of Macrogol.This study is registered with the ClinicalTrials.gov Registry of Clinical Trials, No. NCT02321462

A Novel Approach to Bacterial Expression and Purification of Myristoylated Forms of Neuronal Calcium Sensor Proteins

N-terminal myristoylation is a common co-and post-translational modification of numerous eukaryotic and viral proteins, which affects their interaction with lipids and partner proteins, thereby modulating various cellular processes. Among those are neuronal calcium sensor (NCS) proteins, mediating transduction of calcium signals in a wide range of regulatory cascades, including reception, neurotransmission, neuronal growth and survival. The details of NCSs functioning are of special interest due to their involvement in the progression of ophthalmological and neurodegenerative diseases and their role in cancer. The well-established procedures for preparation of native-like myristoylated forms of recombinant NCSs via their bacterial co-expression with N-myristoyl transferase from Saccharomyces cerevisiae often yield a mixture of the myristoylated and non-myristoylated forms. Here, we report a novel approach to preparation of several NCSs, including recoverin, GCAP1, GCAP2, neurocalcin δ and NCS-1, ensuring their nearly complete N-myristoylation. The optimized bacterial expression and myristoylation of the NCSs is followed by a set of procedures for separation of their myristoylated and non-myristoylated forms using a combination of hydrophobic interaction chromatography steps. We demonstrate that the refolded and further purified myristoylated NCS-1 maintains its Ca2+-binding ability and stability of tertiary structure. The developed approach is generally suited for preparation of other myristoylated proteins

Membrane Binding of Neuronal Calcium Sensor-1: Highly Specific Interaction with Phosphatidylinositol-3-Phosphate

Neuronal calcium sensors are a family of N-terminally myristoylated membrane-binding proteins possessing a different intracellular localization and thereby targeting unique signaling partner(s). Apart from the myristoyl group, the membrane attachment of these proteins may be modulated by their N-terminal positively charged residues responsible for specific recognition of the membrane components. Here, we examined the interaction of neuronal calcium sensor-1 (NCS-1) with natural membranes of different lipid composition as well as individual phospholipids in form of multilamellar liposomes or immobilized monolayers and characterized the role of myristoyl group and N-terminal lysine residues in membrane binding and phospholipid preference of the protein. NCS-1 binds to photoreceptor and hippocampal membranes in a Ca2+-independent manner and the binding is attenuated in the absence of myristoyl group. Meanwhile, the interaction with photoreceptor membranes is less dependent on myristoylation and more sensitive to replacement of K3, K7, and/or K9 of NCS-1 by glutamic acid, reflecting affinity of the protein to negatively charged phospholipids. Consistently, among the major phospholipids, NCS-1 preferentially interacts with phosphatidylserine and phosphatidylinositol with micromolar affinity and the interaction with the former is inhibited upon mutating of N-terminal lysines of the protein. Remarkably, NCS-1 demonstrates pronounced specific binding to phosphoinositides with high preference for phosphatidylinositol-3-phosphate. The binding does not depend on myristoylation and, unexpectedly, is not sensitive to the charge inversion mutations. Instead, phosphatidylinositol-3-phosphate can be recognized by a specific site located in the N-terminal region of the protein. These data provide important novel insights into the general mechanism of membrane binding of NCS-1 and its targeting to specific phospholipids ensuring involvement of the protein in phosphoinositide-regulated signaling pathways

Redox Regulation of Signaling Complex between Caveolin-1 and Neuronal Calcium Sensor Recoverin

Caveolin-1 is a cholesterol-binding scaffold protein, which is localized in detergent-resistant membrane (DRM) rafts and interacts with components of signal transduction systems, including visual cascade. Among these components are neuronal calcium sensors (NCSs), some of which are redox-sensitive proteins that respond to calcium signals by modulating the activity of multiple intracellular targets. Here, we report that the formation of the caveolin-1 complex with recoverin, a photoreceptor NCS serving as the membrane-binding regulator of rhodopsin kinase (GRK1), is a redox-dependent process. Biochemical and biophysical in vitro experiments revealed a two-fold decreased affinity of recoverin to caveolin-1 mutant Y14E mimicking its oxidative stress-induced phosphorylation of the scaffold protein. At the same time, wild-type caveolin-1 demonstrated a 5–10-fold increased affinity to disulfide dimer of recoverin (dRec) or its thiol oxidation mimicking the C39D mutant. The formation of dRec in vitro was not affected by caveolin-1 but was significantly potentiated by zinc, the well-known mediator of redox homeostasis. In the MDCK cell model, oxidative stress indeed triggered Y14 phosphorylation of caveolin-1 and disulfide dimerization of recoverin. Notably, oxidative conditions promoted the accumulation of phosphorylated caveolin-1 in the plasma membrane and the recruitment of recoverin to the same sites. Co-localization of these proteins was preserved upon depletion of intracellular calcium, i.e., under conditions reducing membrane affinity of recoverin but favoring its interaction with caveolin-1. Taken together, these data suggest redox regulation of the signaling complex between recoverin and caveolin-1. During oxidative stress, the high-affinity interaction of thiol-oxidized recoverin with caveolin-1/DRMs may disturb the light-induced translocation of the former within photoreceptors and affect rhodopsin desensitization