Effect of Trace Metal Ions on the Conformational Stability of the Visual Photoreceptor Rhodopsin.

Trace metals are essential elements that play key roles in a number of biochemical processes governing human visual physiology in health and disease. Several trace metals, such as zinc, have been shown to play important roles in the visual phototransduction process. In spite of this, there has been little research conducted on the direct effect of trace metal elements on the visual photoreceptor rhodopsin. In the current study, we have determined the effect of several metal ions, such as iron, copper, chromium, manganese, and nickel, on the conformational stability of rhodopsin. To this aim, we analyzed, by means of UV-visible and fluorescence spectroscopic methods, the effects of these trace elements on the thermal stability of dark rhodopsin, the stability of its active Metarhodopsin II conformation, and its chromophore regeneration. Our results show that copper prevented rhodopsin regeneration and slowed down the retinal release process after illumination. In turn, Fe3+, but not Fe2+, increased the thermal stability of the dark inactive conformation of rhodopsin, whereas copper ions markedly decreased it. These findings stress the important role of trace metals in retinal physiology at the photoreceptor level and may be useful for the development of novel therapeutic strategies to treat retinal disease.

Bio-Based Nano-Enabled Cosmetic Formulations for the Treatment of Cutibacterium acnes-Associated Skin Infections.

Acne is a common chronic skin condition with serious physical and psychosocial consequences. In some cases, the appearance of pimples, whiteheads, or blackheads on the face, neck, and back may lead to scarring, disfiguring, depression, frustration, and anxiety in patients. Current treatments rely on antibiotics to eradicate Cutibacterium acnes (C. acnes), the bacterium responsible for this skin condition. However, these approaches do not scavenge the reactive oxidative species (ROS) generated during disease development and raise concerns about the increase in antimicrobial resistance. In this study, an environmentally friendly and cost-effective self-assembly nanoencapsulation technology based on zein, a bio-based hydrophobic protein, was employed to produce multifunctional essential oil (EO)-loaded nanocapsules (NCs) with superior antioxidant and bactericidal activity toward C. acnes. The NCs displayed "smart" release of the active cargo only under the conditions that were conducive to acne proliferation on skin. Once incorporated into creams, the EO-loaded NCs led to a complete inhibition of C. acnes and demonstrated the capacity to scavenge ROS, thus preventing damage to human skin cells. The in vitro permeation studies revealed that the nanoformulated EO was able to penetrate through the epidermis, indicating its potential for the treatment of skin diseases, such as acne.

Antibody-Enabled Antimicrobial Nanocapsules for Selective Elimination of Staphylococcus aureus.

Targeted bactericide nanosystems hold significant promise to improve the efficacy of existing antimicrobials for treatment of severe bacterial infections, minimizing the side effects and lowering the risk of the development of antibiotic resistance. In this work, we developed antibody-functionalized nanocapsules (NCs) containing antibacterial essential oil (EO) for selective and effective eradication of Staphylococcus aureus. Antibacterial EO NCs were produced via self-assembly nanoencapsulation in the plant-derived protein zein. The obtained EO NCs were decorated with aminocellulose to provide more reactive surface groups for carboxyl-to-amine immobilization of a antibody that is specific against S. aureus. The antibody-enabled EO NCs (Ab@EO NCs) demonstrated 2-fold higher bactericidal efficacy against the targeted bacterium compared to the pristine EO NCs at the same concentrations. The improved antibacterial effect of the Ab@EO NCs toward S. aureus was also confirmed in a real-time assay by monitoring bacterial cells elimination using a quartz crystal microbalance. Furthermore, the Ab@EO NCs selectively decreased the load and changed the cell morphology of the targeted S. aureus in a mixed inoculum with nontargeted Pseudomonas aeruginosa. Applying the nanoformulated antibacterial actives to an in vitro coculture model of the bacteria and skin fibroblasts resulted in suppression of S. aureus growth while preserving the human cells viability. The novel antibody-enabled antibacterial NCs showed potential for improving the treatment efficacy of staphylococcal infections, minimally affecting the beneficial microbial and human cells.

Human Blue Cone Opsin Regeneration Involves Secondary Retinal Binding with Analog Specificity.

Human color vision is mediated by the red, green, and blue cone visual pigments. Cone opsins are G-protein-coupled receptors consisting of an opsin apoprotein covalently linked to the 11-cis-retinal chromophore. All visual pigments share a common evolutionary origin, and red and green cone opsins exhibit a higher homology, whereas blue cone opsin shows more resemblance to the dim light receptor rhodopsin. Here we show that chromophore regeneration in photoactivated blue cone opsin exhibits intermediate transient conformations and a secondary retinoid binding event with slower binding kinetics. We also detected a fine-tuning of the conformational change in the photoactivated blue cone opsin binding site that alters the retinal isomer binding specificity. Furthermore, the molecular models of active and inactive blue cone opsins show specific molecular interactions in the retinal binding site that are not present in other opsins. These findings highlight the differential conformational versatility of human cone opsin pigments in the chromophore regeneration process, particularly compared to rhodopsin, and point to relevant functional, unexpected roles other than spectral tuning for the cone visual pigments.

Strategies to prevent the occurrence of resistance against antibiotics by using advanced materials.

Drug resistance occurrence is a global healthcare concern responsible for the increased morbidity and mortality in hospitals, time of hospitalisation and huge financial loss. The failure of the most antibiotics to kill "superbugs" poses the urgent need to develop innovative strategies aimed at not only controlling bacterial infection but also the spread of resistance. The prevention of pathogen host invasion by inhibiting bacterial virulence and biofilm formation, and the utilisation of bactericidal agents with different mode of action than classic antibiotics are the two most promising new alternative strategies to overcome antibiotic resistance. Based on these novel approaches, researchers are developing different advanced materials (nanoparticles, hydrogels and surface coatings) with novel antimicrobial properties. In this review, we summarise the recent advances in terms of engineered materials to prevent bacteria-resistant infections according to the antimicrobial strategies underlying their design.

Flavonoid allosteric modulation of mutated visual rhodopsin associated with retinitis pigmentosa.

Dietary flavonoids exhibit many biologically-relevant functions and can potentially have beneficial effects in the treatment of pathological conditions. In spite of its well known antioxidant properties, scarce structural information is available on the interaction of flavonoids with membrane receptors. Advances in the structural biology of a specific class of membrane receptors, the G protein-coupled receptors, have significantly increased our understanding of drug action and paved the way for developing improved therapeutic approaches. We have analyzed the effect of the flavonoid quercetin on the conformation, stability and function of the G protein-coupled receptor rhodopsin, and the G90V mutant associated with the retinal degenerative disease retinitis pigmentosa. By using a combination of experimental and computational methods, we suggest that quercetin can act as an allosteric modulator of opsin regenerated with 9-cis-retinal and more importantly, that this binding has a positive effect on the stability and conformational properties of the G90V mutant associated with retinitis pigmentosa. These results open new possibilities to use quercetin and other flavonoids, in combination with specific retinoids like 9-cis-retinal, for the treatment of retinal degeneration associated with retinitis pigmentosa. Moreover, the use of flavonoids as allosteric modulators may also be applicable to other members of the G protein-coupled receptors superfamily.

Structural and functional alterations associated with deutan N94K and R330Q mutations of green cone opsin.

Deuteranopia is an X-linked congenital dichromatic condition in which single point mutations in green cone opsin lead to defective non-functional cone photoreceptor cells. Green cone opsin belongs to the G protein-coupled receptor superfamily and consists of a seven transmembrane helical apoprotein covalently bound to 11-cis-retinal, by means of a protonated Schiff base linkage, in its inactive dark state. Several point mutations in green cone opsin have been reported to cause deuteranopia, but the structural details underlying the molecular mechanisms behind the malfunction of mutated opsins have not been clearly established. Here, deutan N94K and R330Q mutants were studied by introducing these substitutions into the native green cone opsin gene by site-directed mutagenesis. The mutant proteins were purified and analyzed using UV-vis spectroscopy and transducin activation assay. We find that the N94K mutant binds the retinal chromophore by means of an unprotonated Schiff base linkage in contrast to previous studies that reported no chromophore regeneration. The other mutant studied, R330Q, showed impaired functionality as measured by its reduced transducin activation ability when compared to wild-type green cone opsin. A double Cys mutant that could form a stabilizing disulfide bond was used in an attempt to address the instability of the green opsin mutants. Our results suggest the presence of key intramolecular networks which may be disrupted in deuteranopia, and these findings could help in finding therapeutic solutions for treating color blindness. Furthermore, our results can also have implications for the study of other visual pigments and other rhodopsin-like G protein-coupled receptors.

Docosahexaenoic acid phospholipid differentially modulates the conformation of G90V and N55K rhodopsin mutants associated with retinitis pigmentosa.

Rhodopsin is the visual photoreceptor of the retinal rod cells that mediates dim light vision and a prototypical member of the G protein-coupled receptor superfamily. The structural stability and functional performance of rhodopsin are modulated by membrane lipids. Docosahexaenoic acid has been shown to interact with native rhodopsin but no direct evidence has been established on the effect of such lipid on the stability and regeneration of rhodopsin mutants associated with retinal diseases. The stability and regeneration of two thermosensitive mutants G90V and N55K, associated with the retinal degenerative disease retinitis pigmentosa, have been analyzed in docosohexaenoic phospholipid (1,2-didocosa-hexaenoyl-sn-glycero-3-phosphocholine; DDHA-PC) liposomes. G90V mutant reconstituted in DDHA-PC liposomes significantly increased its thermal stability, but N55K mutant showed similar thermal sensitivity both in dodecyl maltoside detergent solution and in DDHA-PC liposomes. The retinal release process, measured by fluorescence spectroscopy, became faster in the lipid system for the two mutants. The opsin conformation was stabilized for the G90V mutant allowing improved retinal uptake whereas no chromophore binding could be detected for N55K opsin after photoactivation. The results emphasize the distinct role of DHA on different phenotypic rhodopsin mutations associated with classical (G90V) and sector (N55K) retinitis pigmentosa.

Innovative Approaches for Controlling Clinically Relevant Biofilms: Current Trends and Future Prospects.

Bacteria that colonize and form biofilms on living tissues and medical devices are a global healthcare concern. They cause life threatening infections and are associated with increased mortality and morbidity in the hospitals. Although antibiotics have been successfully applied for treatment of bacterial diseases, the adaptive and genetic changes of the microorganisms within the biofilms make them inherently resistant to all known antibacterial agents. Therefore, novel antimicrobial strategies that do not exert selective pressure on bacterial population and minimize the risk of resistance occurrence have been sought to prevent and treat biofilm related infections. A critical overview of the numerous groups and the rationale of advanced materials and surfaces with antibacterial and antibiofilm properties is the aim of this review. The development of antibiofilm coatings based on molecules interfering with bacterial cell-to-cell communication and biofilm integrity are discussed. Nano-scale transformation of obsolete antibiotics and surface functionalization with bacteriophages and natural antibacterials including enzymes, antimicrobial peptides, and polyphenols are also considered. Finally, recent efforts to design new generation of integrated antibacterial materials are reported.

Functional role of positively selected amino acid substitutions in mammalian rhodopsin evolution.

Visual rhodopsins are membrane proteins that function as light photoreceptors in the vertebrate retina. Specific amino acids have been positively selected in visual pigments during mammal evolution, which, as products of adaptive selection, would be at the base of important functional innovations. We have analyzed the top candidates for positive selection at the specific amino acids and the corresponding reverse changes (F13M, Q225R and A346S) in order to unravel the structural and functional consequences of these important sites in rhodopsin evolution. We have constructed, expressed and immunopurified the corresponding mutated pigments and analyzed their molecular phenotypes. We find that position 13 is very important for the folding of the receptor and also for proper protein glycosylation. Position 225 appears to be important for the function of the protein affecting the G-protein activation process, and position 346 would also regulate functionality of the receptor by enhancing G-protein activation and presumably affecting protein phosphorylation by rhodopsin kinase. Our results represent a link between the evolutionary analysis, which pinpoints the specific amino acid positions in the adaptive process, and the structural and functional analysis, closer to the phenotype, making biochemical sense of specific selected genetic sequences in rhodopsin evolution.

Beyond spectral tuning: human cone visual pigments adopt different transient conformations for chromophore regeneration.

Human red and green visual pigments are seven transmembrane receptors of cone photoreceptor cells of the retina that mediate color vision. These pigments share a very high degree of homology and have been assumed to feature analogous structural and functional properties. We report on a different regeneration mechanism among red and green cone opsins with retinal analogs using UV-Vis/fluorescence spectroscopic analyses, molecular modeling and site-directed mutagenesis. We find that photoactivated green cone opsin adopts a transient conformation which regenerates via an unprotonated Schiff base linkage with its natural chromophore, whereas red cone opsin forms a typical protonated Schiff base. The chromophore regeneration kinetics is consistent with a secondary retinal uptake by the cone pigments. Overall, our findings reveal, for the first time, structural differences in the photoactivated conformation between red and green cone pigments that may be linked to their molecular evolution, and support the proposal of secondary retinal binding to visual pigments, in addition to binding to the canonical primary site, which may serve as a regulatory mechanism of dark adaptation in the phototransduction process.

Zinc Is Involved in Depression by Modulating G Protein-Coupled Receptor Heterodimerization.

5-Hydroxytryptamine 1A receptor and galanin receptor 1 belong to the G protein-coupled receptors superfamily, and they have been described to heterodimerize triggering an anomalous physiological state that would underlie depression. Zinc supplementation has been widely reported to improve treatment against major depressive disorder. Our work has focused on the study and characterization of these receptors and its relationships with zinc both under purified conditions and in cell culture. To this aim, we have designed a strategy to purify the receptors in a conformationally active state. We have used receptors tagged with the monoclonal Rho-1D4 antibody and employed ligand-assisted purification in order to successfully purify both receptors in a properly folded and active state. The interaction between both purified receptors has been analyzed by surface plasmon resonance in order to determine the kinetics of dimerization. Zinc effect on heteromer has also been tested using the same methodology but exposing the 5-hydroxytryptamine 1A receptor to zinc before the binding experiment. These results, combined with Förster resonance energy transfer (FRET) measurements, in the absence and presence of zinc, suggest that this ion is capable of disrupting this interaction. Moreover, molecular modeling suggests that there is a coincidence between zinc-binding sites and heterodimerization interfaces for the serotonin receptor. Our results establish a rational explanation for the role of zinc in the molecular processes associated with receptor-receptor interactions and its relationship with depression, in agreement with previously reported evidence for the positive effects of zinc in depression treatment, and the involvement of our target dimer in the same disease.

The zinc binding receptor GPR39 interacts with 5-HT1A and GalR1 to form dynamic heteroreceptor complexes with signaling diversity.

GPR39 is a class A G protein-coupled receptor involved in zinc binding and glucose homeostasis regulation, among other physiological processes. GPR39 was originally thought to be the receptor for obestatin peptide but this view has been challenged. However, activation of this receptor by zinc has been clearly established. Recent studies suggest that low GPR39 expression, due to deficient zinc levels, is involved in major depressive disorder. We have previously reported that zinc can alter receptor-receptor interactions and favor specific receptor interactions. In order to unravel the effect of zinc on specific G protein-coupled receptor association processes, we have performed FRET and co-immunopurification studies with GPR39 and 5-HT1A and GalR1 which have been shown to dimerize. Our results suggest that zinc can modulate the formation of specific 5-HT1A-GPR39 and GalR1-5-HT1A-GPR39 heteroreceptor complexes under our experimental conditions. We have analyzed the differences in signaling between the mono-homomeric receptors 5-HT1A, GalR1 and GPR39 and the heteroreceptor complexes between them Our results show that the GPR39-5-HT1A heterocomplex has additive functionalities when compared to the monomeric-homomeric receptors upon receptor activation. In addition, the heterocomplex including also GalR1 shows a different behavior, upon exposure to the same agonists. Furthermore, these processes appear to be regulated by zinc. These findings provide a rationale for the antidepressive effect widely described for zinc because pro-depressive heterocomplexes are predominant at low zinc concentration levels.

Phospholipid Bicelles Improve the Conformational Stability of Rhodopsin Mutants Associated with Retinitis Pigmentosa.

Mutations in the visual photoreceptor rhodopsin are the cause of the retinal degenerative disease retinitis pigmentosa. Some naturally occurring mutations can lead to protein conformational instability. Two such mutations, N55K and G90V, in the first and second transmembrane helices of the receptor, have been associated with sector and classical retinitis pigmentosa, respectively, and showed enhanced thermal sensitivity. We have carefully analyzed the effect of phospholipid bicelles on the stability and ligand binding properties of these two mutants and compared it with those of the detergent-solubilized samples. We have used a phospholipid bilayer consisting of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC). We find that DMPC/DHPC bicelles dramatically increase the thermal stability of the rhodopsin mutants G90V and N55K. The chromophore stability and regeneration of the mutants were also increased in bicelles when compared to their behavior in a dodecyl maltoside detergent solution. The retinal release process was slowed in bicelles, and chromophore entry, after illumination, was improved for the G90V mutant but not for N55K. Furthermore, fluorescence spectroscopy measurements showed that bicelles allowed more exogenous retinal binding to the photoactivated G90V mutant than in a detergent solution. In contrast, N55K could not reposition any chromophore either in the detergent or in bicelles. The results demonstrate that DMPC/DHPC bicelles can counteract the destabilizing effect of the disease-causing mutations and can modulate the structural changes in the ensuing receptor photoactivation in a distinct specific manner for different retinitis pigmentosa mutant phenotypes.

Differential light-induced responses in sectorial inherited retinal degeneration.

Retinitis pigmentosa (RP) is a group of genetically and clinically heterogeneous inherited degenerative retinopathies caused by abnormalities of photoreceptors or retinal pigment epithelium in the retina leading to progressive sight loss. Rhodopsin is the prototypical G-protein-coupled receptor located in the vertebrate retina and is responsible for dim light vision. Here, novel M39R and N55K variants were identified as causing an intriguing sector phenotype of RP in affected patients, with selective degeneration in the inferior retina. To gain insights into the molecular aspects associated with this sector RP phenotype, whose molecular mechanism remains elusive, the mutations were constructed by site-directed mutagenesis, expressed in heterologous systems, and studied by biochemical, spectroscopic, and functional assays. M39R and N55K opsins had variable degrees of chromophore regeneration when compared with WT opsin but showed no gross structural misfolding or altered trafficking. M39R showed a faster rate for transducin activation than WT rhodopsin with a faster metarhodopsinII decay, whereas N55K presented a reduced activation rate and an altered photobleaching pattern. N55K also showed an altered retinal release from the opsin binding pocket upon light exposure, affecting its optimal functional response. Our data suggest that these sector RP mutations cause different protein phenotypes that may be related to their different clinical progression. Overall, these findings illuminate the molecular mechanisms of sector RP associated with rhodopsin mutations.

G-protein-coupled receptors oligomerization: emerging signaling units and new opportunities for drug design.

G-protein-coupled receptors (GPCRs) are a widespread family of transmembrane receptors with different physiologically relevant functions. Alterations in the structure and function of these receptors at different levels (ligand binding, signaling and trafficking) may result in a number of pathological conditions which represent a major health problem. Mutations in these receptors are also linked to different inherited diseases for which there is no cure to date. Rationale design, based on receptor structural knowledge, is needed for the discovery of novel drugs with higher selectivity and less side effects. In fact, about 50% of the drugs currently under development target this kind of receptors. Oligomerization among GPCRs has been clearly established from experimental, particularly in vitro, studies. Moreover, homo and heterodimerization provide new unexpected clues for explaining the molecular mechanisms underlying some diseases in which GPCRs signaling might be affected. In this review we will analyze GPCRs structure and function for a better understanding of the dimerization process and the experimental approaches currently used to detect such interactions. Furthermore, how drugs targeting heteromers can represent new opportunities to tackle novel and safer treatments of some pathologies will be described. Recent results, in this regard, will be reported as encouraging examples in the field. Finally, the newest technologies available for developing drugs targeting heteromers will also be reviewed highlighting the importance of bivalent ligands that emerge as very powerful molecules interacting with heteromers.

Mercury-induced dark-state instability and photobleaching alterations of the visual g-protein coupled receptor rhodopsin.

Mercuric compounds were previously shown to affect the visual phototransduction cascade, and this could result in vision impairment. We have analyzed the effect of mercuric chloride on the structure and stability of the dim light vision photoreceptor rhodopsin. For this purpose, we have used both native rhodopsin immunopurified from bovine retinas and a recombinant mutant rhodopsin carrying several Cys to Ser substitutions in order to investigate the potential binding site of mercury on the receptor. Our results show that mercuric chloride dramatically reduces the stability of dark-state rhodopsin and alters the molecular features of the photoactived conformation obtained upon illumination by eliciting the formation of an altered photointermediate. The thermal bleaching kinetics of native and mutant rhodopsin is markedly accelerated by mercury in a concentration-dependent manner, and its chromophore regeneration ability is severely reduced without significantly affecting its G-protein activation capacity. Furthermore, fluorescence spectroscopic measurements on the retinal release process, ensuing illumination, suggest that mercury impairs complete retinal release from the receptor binding pocket. Our results provide further support for the capacity of mercury as a hazardous metal ion with reported deleterious effect on vision and provide a molecular explanation for such an effect at the rhodopsin photoreceptor level. We suggest that mercury could alter vision by acting in a specific manner on the molecular components of the retinoid cycle, particularly by modifying the ability of the visual photoreceptor protein rhodopsin to be regenerated and to be normally photoactivated by light.

Binding specificity of retinal analogs to photoactivated visual pigments suggest mechanism for fine-tuning GPCR-ligand interactions.

11-cis-retinal acts as an inverse agonist stabilizing the inactive conformation of visual pigments, and upon photoactivation, it isomerizes to all-trans-retinal, initiating signal transduction. We have analyzed opsin regeneration with retinal analogs for rhodopsin and red cone opsin. We find differential binding of the analogs to the receptors after photobleaching and a dependence of the binding kinetics on the oligomerization state of the protein. The results outline the sensitivity of retinal entry to the binding pocket of visual receptors to the specific conformation adopted by the receptor and by the molecular architecture defined by specific amino acids in the binding pocket and the retinal entry site, as well as the topology of the retinal analog. Overall, our findings highlight the specificity of the ligand-opsin interactions, a feature that can be shared by other G-protein-coupled receptors.

Evaluación de la enseñanza sobre violencia en la Facultad de Medicina / Evaluation of teaching on violence at a Faculty of Medicine

Objetivo: Evaluar los cambios e innovaciones alcanzados en la enseñanza sobre violencia en la Facultad de Medicina, Universidad Nacional Mayor de San Marcos (UNMSM) en el lapso de 2005 al 2011, luego de realizar acciones de coordinación y sensibilización con las Escuelas Académico Profesionales (EAP) y Departamentos Académicos (DA) para efectuar cambios curriculares. Diseño: estudio descriptivo, tranversal, evaluativo de análisis de sílabos. Institución: Facultad de Medicina, UNMSM, Lima, Perú. Material: Sílabos. Metodología: En el año 2012 se efectuó un estudio de 79 sílabos de asignaturas seleccionadas de las 5 Escuelas Académico Profesionales (EAP) de la Facultad de Medicina, del año académico 2011. Principales medidas de resultados: Cambios e innovaciones de contenidos sobre violencia. Resultados: Se han incrementado los contenidos sobre violencia en las asignaturas seleccionadas, de 30/81 (37%) en el año 2005 a 58/79 (73,4%) en el año 2011. En la EAP de Medicina mejoró de 47% a 89,4%, en la EAP de Obstetricia de 45% a 85%, en la EAP de Enfermería de 40,6% a 72,7%, en la EAP de Tecnología Médica de 31,5% a 42,1% y en la EAP de Nutrición de 10% a 50%. Conclusiones: La enseñanza sobre violencia en la Facultad de Medicina mejoró significativamente del año 2005 al 2011, lo que contribuirá a una mejor formación profesional en la Facultad de Medicina para la atención de las víctimas de la violencia y para contribuir a la solución de este grave problema nacional de salud.

Objectives: To determine changes and innovations in teaching on violence at the Faculty of Medicine, Universidad Nacional Mayor de San Marcos (UNMSM), Lima, Peru, from 2005 until academic year 2011, time in which diverse coordinations with academic and faculty authorities were done to improve teaching on violence. Design: Descriptive, transversal and analytical study of syllabi. Institution: Faculty of Medicine, UNMSM, Lima, Peru. Material: Syllabi. Methodology: In year 2012, 79 selected syllabi corresponding to academic year 2011 of the five schools of the Faculty of Medicine were studied. Main outcome measures: Changes and innovations in violence contents. Results: There was increment in contents on teaching of violence in selected syllabi from 30/81 (37%) in year 2005 to 58/79 (73.4%) in year 2011. In the School of Medicine increase was from 47% to 89.4%, in the School of Midwifery from 45% to 85%, in the School of Nursery from 40.6% to 72.7%, in the School of Medical Technology from 31.5% to 47.3%, and in the School of Nutrition from 10% to 50%. Conclusions: Teaching on violence has improved from year 2005 to 2011 at the Faculty of Medicine; therefore professionals trained by the Faculty of Medicine will have better skills to attend violence victims and will contribute to the solution of this public health problem.

Improved conformational stability of the visual G protein-coupled receptor rhodopsin by specific interaction with docosahexaenoic acid phospholipid.

Rhodopsin is the photoreceptor located in the rod cells of the retina. It has seven transmembrane helices and is a prototypic member of the G protein-coupled receptor superfamily. The structures and functions of these receptors are clearly affected by the lipid composition of the cell membrane, and their study in a purified recombinant form is usually performed in detergent solution. There is a need to study these receptors in a physiologically relevant environment because the lipid environment is known to have an important effect on their function. In this work, rhodopsin reconstituted in docosahexaenoic acid (DHA) liposomes is shown to have more thermal stability than when it is solubilised with the neutral detergent dodecyl maltoside. Moreover, the specific interaction between rhodopsin and DHA was followed by means of Langmuir experiments with insertion of rhodopsin into lipid monolayers; this showed high affinity for the lipid-receptor interaction. Furthermore, fluorescence spectroscopy measurements indicate that the conformation of opsin obtained after photobleaching is preserved in DHA-containing liposomes, thereby allowing retinal to re-enter the binding pocket even long after bleaching. Overall, our results demonstrate that liposomes of this specific lipid provide a more stable environment for ground-state inactive rhodopsin in the dark, than dodecyl maltoside detergent, and that this lipid can also preserve the native correctly folded ligand-free opsin conformation obtained after illumination. This strategy will be used in further studies on mutations of rhodopsin associated with congenital retinopathies.