The rise and shine of yeast optogenetics.

Optogenetics refers to the control of biological processes with light. The activation of cellular phenomena by defined wavelengths has several advantages compared with traditional chemically inducible systems, such as spatiotemporal resolution, dose-response regulation, low cost, and moderate toxic effects. Optogenetics has been successfully implemented in yeast, a remarkable biological platform that is not only a model organism for cellular and molecular biology studies, but also a microorganism with diverse biotechnological applications. In this review, we summarize the main optogenetic systems implemented in the budding yeast Saccharomyces cerevisiae, which allow orthogonal control (by light) of gene expression, protein subcellular localization, reconstitution of protein activity, and protein sequestration by oligomerization. Furthermore, we review the application of optogenetic systems in the control of metabolic pathways, heterologous protein production and flocculation. We then revise an example of a previously described yeast optogenetic switch, named FUN-LOV, which allows precise and strong activation of the target gene. Finally, we describe optogenetic systems that have not yet been implemented in yeast, which could therefore be used to expand the panel of available tools in this biological chassis. In conclusion, a wide repertoire of optogenetic systems can be used to address fundamental biological questions and broaden the biotechnological toolkit in yeast.

Modular and Molecular Optimization of a LOV (Light-Oxygen-Voltage)-Based Optogenetic Switch in Yeast.

Optogenetic switches allow light-controlled gene expression with reversible and spatiotemporal resolution. In Saccharomyces cerevisiae, optogenetic tools hold great potential for a variety of metabolic engineering and biotechnology applications. In this work, we report on the modular optimization of the fungal light-oxygen-voltage (FUN-LOV) system, an optogenetic switch based on photoreceptors from the fungus Neurospora crassa. We also describe new switch variants obtained by replacing the Gal4 DNA-binding domain (DBD) of FUN-LOV with nine different DBDs from yeast transcription factors of the zinc cluster family. Among the tested modules, the variant carrying the Hap1p DBD, which we call "HAP-LOV", displayed higher levels of luciferase expression upon induction compared to FUN-LOV. Further, the combination of the Hap1p DBD with either p65 or VP16 activation domains also resulted in higher levels of reporter expression compared to the original switch. Finally, we assessed the effects of the plasmid copy number and promoter strength controlling the expression of the FUN-LOV and HAP-LOV components, and observed that when low-copy plasmids and strong promoters were used, a stronger response was achieved in both systems. Altogether, we describe a new set of blue-light optogenetic switches carrying different protein modules, which expands the available suite of optogenetic tools in yeast and can additionally be applied to other systems.

[Methemoglobinemia caused by dapsone. Report of one case]. / Metahemoglobinemia, una entidad de diagnóstico complejo. Reporte de un caso.

Methemoglobinemia is a rare condition with serious consequences if not diagnosed. We report the case of a 64-year-old woman with a history of allergy to sulfa drugs and a recent diagnosis of a small vessel vasculitis (ANCA-p) who started induction therapy with corticosteroids and rituximab. Due to the need for infectious prophylaxis, and considering her history, dapsone was administered instead of cotrimoxazole after ruling out glucose-6-phosphate dehydrogenase deficiency. During the admission to the hospital for her second dose of rituximab, and while being asymptomatic, she persistently presented a pulse oximetry ≪ 90% despite the administration of O2. Therefore, the infusion was postponed to study the patient. The arterial gasometric study by direct potentiometry revealed an O2 saturation of 98%, with a saturation gap > 5%. Considering the use of dapsone, a methemoglobinemia was suspected and confirmed by co-oximetry (methemoglobinemia 9%). Dapsone was suspended and one week later, her methemoglobinemia was absent.

Optogenetic switches for light-controlled gene expression in yeast.

Light is increasingly recognized as an efficient means of controlling diverse biological processes with high spatiotemporal resolution. Optogenetic switches are molecular devices for regulating light-controlled gene expression, protein localization, signal transduction and protein-protein interactions. Such molecular components have been mainly developed through the use of photoreceptors, which upon light stimulation undergo conformational changes passing to an active state. The current repertoires of optogenetic switches include red, blue and UV-B light photoreceptors and have been implemented in a broad spectrum of biological platforms. In this review, we revisit different optogenetic switches that have been used in diverse biological platforms, with emphasis on those used for light-controlled gene expression in the budding yeast Saccharomyces cerevisiae. The implementation of these switches overcomes the use of traditional chemical inducers, allowing precise control of gene expression at lower costs, without leaving chemical traces, and positively impacting the production of high-value metabolites and heterologous proteins. Additionally, we highlight the potential of utilizing this technology beyond laboratory strains, by optimizing it for use in yeasts tamed for industrial processes. Finally, we discuss how fungal photoreceptors could serve as a source of biological parts for the development of novel optogenetic switches with improved characteristics. Although optogenetic tools have had a strong impact on basic research, their use in applied sciences is still undervalued. Therefore, the invitation for the future is to utilize this technology in biotechnological and industrial settings.

Coupling Cell Communication and Optogenetics: Implementation of a Light-Inducible Intercellular System in Yeast.

Cell communication is a widespread mechanism in biology, allowing the transmission of information about environmental conditions. In order to understand how cell communication modulates relevant biological processes such as survival, division, differentiation, and apoptosis, different synthetic systems based on chemical induction have been successfully developed. In this work, we coupled cell communication and optogenetics in the budding yeast Saccharomyces cerevisiae. Our approach is based on two strains connected by the light-dependent production of α-factor pheromone in one cell type, which induces gene expression in the other type. After the individual characterization of the different variants of both strains, the optogenetic intercellular system was evaluated by combining the cells under contrasting illumination conditions. Using luciferase as a reporter gene, specific co-cultures at a 1:1 ratio displayed activation of the response upon constant blue light, which was not observed for the same cell mixtures grown in darkness. Then, the system was assessed at several dark/blue-light transitions, where the response level varies depending on the moment in which illumination was delivered. Furthermore, we observed that the amplitude of response can be tuned by modifying the initial ratio between both strains. Finally, the two-population system showed higher fold inductions in comparison with autonomous strains. Altogether, these results demonstrated that external light information is propagated through a diffusible signaling molecule to modulate gene expression in a synthetic system involving microbial cells, which will pave the road for studies allowing optogenetic control of population-level dynamics.

Interactions between Core Elements of the Botrytis cinerea Circadian Clock Are Modulated by Light and Different Protein Domains.

Botrytis cinerea possesses a complex light-sensing system composed of eleven photoreceptors. In B. cinerea, bcwcl1 encodes for the BcWCL1 protein, the orthologue of the blue-light photoreceptor WC-1 from Neurospora crassa. The functional partner of BcWCL1 is the BcWCL2 protein, both interacting in the nucleus and forming the B. cinerea white collar complex (BcWCC). This complex is required for photomorphogenesis and circadian regulation. However, no molecular evidence shows a light-dependent interaction between the BcWCC components or light-sensing capabilities in BcWCL1. In this work, by employing a yeast two-hybrid system that allows for the in vivo analysis of protein-protein interactions, we confirm that BcWCL1 and BcWCL2 interact in the absence of light as well as upon blue-light stimulation, primarily through their PAS (Per-Arnt-Sim) domains. Deletion of the PAS domains present in BcWCL1 (BcWCL1PAS∆) or BcWCL2 (BcWCL2PAS∆) severely impairs the interaction between these proteins. Interestingly, the BcWCL1PAS∆ protein shows a blue-light response and interacts with BcWCL2 or BcWCL2PAS∆ upon light stimulation. Finally, we demonstrate that BcWCL1 and BcWCL1PAS∆ respond to blue light by introducing a point mutation in the photoactive cysteine, confirming that both proteins are capable of light sensing. Altogether, the results revealed the complexity of protein-protein interactions occurring between the core elements of the B. cinerea circadian clock.

Effect of an infection control program on the frequency of nosocomial viral respiratory infections.

We determined the rate of nosocomial viral respiratory infection in infants and the effect of an infection control program during 4 winter seasons. The rate of nosocomial viral respiratory infection decreased from 6.09 episodes per 100 patients admitted during the first study year to 1.46 episodes per 100 patients admitted during the last study year.

Fungal Light-Oxygen-Voltage Domains for Optogenetic Control of Gene Expression and Flocculation in Yeast.

Optogenetic switches permit accurate control of gene expression upon light stimulation. These synthetic switches have become a powerful tool for gene regulation, allowing modulation of customized phenotypes, overcoming the obstacles of chemical inducers, and replacing their use by an inexpensive resource: light. In this work, we implemented FUN-LOV, an optogenetic switch based on the photon-regulated interaction of WC-1 and VVD, two LOV (light-oxygen-voltage) blue-light photoreceptors from the fungus Neurospora crassa When tested in yeast, FUN-LOV yields light-controlled gene expression with exquisite temporal resolution and a broad dynamic range of over 1,300-fold, as measured by a luciferase reporter. We also tested the FUN-LOV switch for heterologous protein expression in Saccharomyces cerevisiae, where Western blot analysis confirmed strong induction upon light stimulation, surpassing by 2.5 times the levels achieved with a classic GAL4/galactose chemical-inducible system. Additionally, we utilized FUN-LOV to control the ability of yeast cells to flocculate. Light-controlled expression of the flocculin-encoding gene FLO1, by the FUN-LOV switch, yielded flocculation in light (FIL), whereas the light-controlled expression of the corepressor TUP1 provided flocculation in darkness (FID). Altogether, the results reveal the potential of the FUN-LOV optogenetic switch to control two biotechnologically relevant phenotypes such as heterologous protein expression and flocculation, paving the road for the engineering of new yeast strains for industrial applications. Importantly, FUN-LOV's ability to accurately manipulate gene expression, with a high temporal dynamic range, can be exploited in the analysis of diverse biological processes in various organisms.IMPORTANCE Optogenetic switches are molecular devices which allow the control of different cellular processes by light, such as gene expression, providing a versatile alternative to chemical inducers. Here, we report a novel optogenetic switch (FUN-LOV) based on the LOV domain interaction of two blue-light photoreceptors (WC-1 and VVD) from the fungus N. crassa In yeast cells, FUN-LOV allowed tight regulation of gene expression, with low background in darkness and a highly dynamic and potent control by light. We used FUN-LOV to optogenetically manipulate, in yeast, two biotechnologically relevant phenotypes, heterologous protein expression and flocculation, resulting in strains with potential industrial applications. Importantly, FUN-LOV can be implemented in diverse biological platforms to orthogonally control a multitude of cellular processes.

GPD1 and ADH3 Natural Variants Underlie Glycerol Yield Differences in Wine Fermentation.

Glycerol is one of the most important by-products of alcohol fermentation, and depending on its concentration it can contribute to wine flavor intensity and aroma volatility. Here, we evaluated the potential of utilizing the natural genetic variation of non-coding regions in budding yeast to identify allelic variants that could modulate glycerol phenotype during wine fermentation. For this we utilized four Saccharomyces cerevisiae strains (WE - Wine/European, SA - Sake, NA - North American, and WA - West African), which were previously profiled for genome-wide Allele Specific Expression (ASE) levels. The glycerol yields under Synthetic Wine Must (SWM) fermentations differed significantly between strains; WA produced the highest glycerol yields while SA produced the lowest yields. Subsequently, from our ASE database, we identified two candidate genes involved in alcoholic fermentation pathways, ADH3 and GPD1, exhibiting significant expression differences between strains. A reciprocal hemizygosity assay demonstrated that hemizygotes expressing GPD1WA , GPD1SA , ADH3WA and ADH3SA alleles had significantly greater glycerol yields compared to GPD1WE and ADH3WE . We further analyzed the gene expression profiles for each GPD1 variant under SWM, demonstrating that the expression of GPD1WE occurred earlier and was greater compared to the other alleles. This result indicates that the level, timing, and condition of expression differ between regulatory regions in the various genetic backgrounds. Furthermore, promoter allele swapping demonstrated that these allele expression patterns were transposable across genetic backgrounds; however, glycerol yields did not differ between wild type and modified strains, suggesting a strong trans effect on GPD1 gene expression. In this line, Gpd1 protein levels in parental strains, particularly Gpd1pWE, did not necessarily correlate with gene expression differences, but rather with glycerol yield where low Gpd1pWE levels were detected. This suggests that GPD1WE is influenced by recessive negative post-transcriptional regulation which is absent in the other genetic backgrounds. This dissection of regulatory mechanisms in GPD1 allelic variants demonstrates the potential to exploit natural alleles to improve glycerol production in wine fermentation and highlights the difficulties of trait improvement due to alternative trans-regulation and gene-gene interactions in the different genetic background.

Metahemoglobinemia, una entidad de diagnóstico complejo. Reporte de un caso

Methemoglobinemia is a rare condition with serious consequences if not diagnosed. We report the case of a 64-year-old woman with a history of allergy to sulfa drugs and a recent diagnosis of a small vessel vasculitis (ANCA-p) who started induction therapy with corticosteroids and rituximab. Due to the need for infectious prophylaxis, and considering her history, dapsone was administered instead of cotrimoxazole after ruling out glucose-6-phosphate dehydrogenase deficiency. During the admission to the hospital for her second dose of rituximab, and while being asymptomatic, she persistently presented a pulse oximetry ≪ 90% despite the administration of O2. Therefore, the infusion was postponed to study the patient. The arterial gasometric study by direct potentiometry revealed an O2 saturation of 98%, with a saturation gap > 5%. Considering the use of dapsone, a methemoglobinemia was suspected and confirmed by co-oximetry (methemoglobinemia 9%). Dapsone was suspended and one week later, her methemoglobinemia was absent.

Risk factors for low birth weight: a review.

Low birth weight (LBW) is one of the main predictors of infant mortality. The global incidence of LBW is around 17%, although estimates vary from 19% in the developing countries (countries where it is an important public health problem) to 5-7% in the developed countries. The incidence in Spain in the decade 1980-1989 was about 5.7%. LBW is generally associated with situations in which uterine malnutrition is produced due to alterations in placental circulation. There are many known risk factors, the most important of which are socio-economic factors, medical risks before or during gestation and maternal lifestyles. However, although interventions exist to prevent many of these factors before and during pregnancy, the incidence of LBW has not decreased.

[Vaccine storage cold chain at primary care centers in one area of Madrid: keeping the chain intact and degree of knowledge]. / Cadena del frío para la conservación de las vacunas en los centros de atención primaria de un area de Madrid: mantenimiento y nivel de conocimientos.

BACKGROUND: Vaccines are heat-labile medications, and to guarantee their immunogenicity and safeguarding effectiveness as part of immunization programs, it is absolutely essential that the "Cold Chain" go unbroken. Fundamental thereto is the personnel responsible for the vaccines, who must know the stability-related characteristics of each preparation so as to prevent handling errors. The purpose of this study was that of ascertaining how the cold chain is kept intact in primary care systems in one healthcare area of the Autonomous Community of Madrid, as well as determining the degree of information possessed by those responsible for vaccines as far as their heat-stability is concerned. METHODS: A cross-sectional study has been made at 46 primary care vaccination points. The data was gathered by means of a personal interview by one single researcher. RESULTS: The participation rate was 93.5% (43/46). In all cases, there was a maximum and minimum thermometer and monthly temperature record. An unsuitable temperature was found in three cases (6.97%). The percentage of professionals who were aware of the effect freezing has on vaccines varied greatly: 53.5%, 51.2%, 44.2% and 53.5% for diphtheria-tetanus-pertussis (DTP), hepatitis B (HBV), oral polio (OPV) and measles-mumps-rubella (MMR) respectively. And only 32% were familiar with the shake test. CONCLUSION: The professionals were found to be properly trained regarding the effect which high temperatures have on vaccines, but it is necessary for their training with regard to the instability of adsorbed preparations when frozen must be further strengthened.

Marketing social y su influencia en la solución de problemas de salud / Social marketing and its influence in the solution of health problems

Introducción: El marketing social es una disciplina de la mercadotecnia dirigida a influir en el comportamiento de las personas a fin de que mejoren su bienestar y el de la sociedad. Por sus características tiene potencialidades de influir en la solución de problemas de salud a nivel individual, grupal y social, en especial desde el punto de vista de las acciones de comunicación a aplicarse, como las actividades sociales, campañas sociales, charlas, relaciones públicas, ruedas de prensa, entre otras. Objetivo: El objetivo del artículo es valorar a partir del criterio de expertos, la influencia que puede ejercer el marketing social en la solución de problemas de salud en Ecuador. Métodos: Se aplicó un cuestionario para conocer el criterio de 10 especialistas de las ciencias médicas y administrativas de Ecuador, en función de la influencia que pueden ejercer los instrumentos y estrategias de comunicación de marketing social en la solución de problemas de salud. El procesamiento de los datos se realizó con Microsoft Excel, para determinar el comportamiento descriptivo de los mismos y de forma inferencial a través del Coeficiente R de Pearson. Resultados: A partir del criterio de los expertos se determinó la importancia de las actividades sociales para la solución de problemas de salud en Ecuador, en especial las campañas sociales explicativas y de desmotivación de vicios, así como los mensajes de motivación. Destacan como principales objetos de las campañas las enfermedades epidémicas y del desarrollo, y el gobierno nacional como principal agente de marketing social. Mientras que la comunidad tiene una baja participación y negativas relaciones con otras variables estudiadas. El control del proceso de comunicación limita la efectividad de las acciones de marketing social. Conclusiones: El análisis empírico desarrollado a partir del criterio de expertos permitió diagnosticar la influencia que ejerce el marketing social en la solución de problemas de salud, así como los principales instrumentos de comunicación que se emplean, los agentes más activos y algunas insuficiencias en el proceso(AU)

Introduction: Social marketing is a discipline of marketing aimed at influencing people's behavior in order to improve their well-being and that of society. Because of its characteristics, it has the potential to influence the solution of health problems at the individual, group and social level, especially from the point of view of communication actions to be applied, such as social activities, social campaigns, talks, public relations, press conferences, among others. Objective: The objective of the article is to evaluate, based on the criterion of experts, the influence that social marketing can exert on solving health problems in Ecuador. Methods: A questionnaire was applied to know the criteria of 10 specialists from the medical and administrative sciences of Ecuador, depending on the influence that can be exerted on the instruments and strategies of communication of social marketing in the solution of health problems. The data processing was done with Microsoft Excel, to determine the descriptive behavior of the same and inferential form through Pearson R Coefficient. Results: Based on the criteria of the experts, the importance of social activities for the solution of health problems in Ecuador was determined, especially the explanatory social campaigns and demotivation of vices, as well as motivational messages. The epidemic and developmental diseases are highlighted as the main objects of the campaigns, and the national government is the main agent of social marketing. While the community has low participation and negative relationships with other variables studied. Controlling the communication process limits the effectiveness of social marketing actions. Conclusions: The empirical analysis developed from the expert criterion allowed to diagnose the influence of social marketing in the solution of health problems, as well as the main communication tools used, the most active agents and some shortcomings in the process(AU)

[Analysis of short-term time off of health workers in a health area]. / Análisis de la incapacidad temporal en trabajadores de la rama sanitaria de un área de salud.

OBJECTIVE: To describe the characteristics of short-term time off work (STT): duration, causes and distribution by age, and sex. DESIGN: Descriptive, observational study. SETTING: Area 1, Madrid, Spain. PARTICIPANTS: Health workers with STT in Area 1, Madrid, during the first 6 months of 2003. MAIN MEASUREMENTS: The variables studied were: date of start and finish of STT, its duration, its cause (ICD-9), age, and sex. RESULTS: Four-hundred eighteen health workers (3.7%), with a mean age of 32.7+/-20 years old and 78.79% women. Respiratory pathology was the most frequent cause of STT (38%) in both sexes and in all age-groups, except in the youngest (<25), in whom bone and muscle pathology was more common. In decreasing order of frequency appeared bone and muscle pathology (26%), infections (13%), and mental illness (10%). The average duration of STT was 38.8+/-88.7 days. There was a statistically significant relationship between the duration of STT and its cause. Respiratory pathology was the shortest (8.85; 95% CI, 6.54-11.17) and mental illness, the longest (153.8; 95% CI, 87-220.75). The age group with most STT (37-45 years old) had a mean length of STT lower (26.93; 95% CI, 16.34-37.51) than the oldest age group (>56), which had less, but longer-lasting STT (57.18; 95% CI, 10.74-103.63), though this was not statistically significant. CONCLUSIONS: The pathology causing STT is the factor that most determines STT's duration. Its prevention would be the best way of managing STT.

Cognitive assessment of the non-demented elderly community dwellers in Spain.

OBJECTIVE: The prevalence of cognitive impairment and population normative values for cognitive function were assessed, for the first time, in 2,630 subjects representative of the non-demented community dweller population > or =65 years in Spain, a southern European country with a sizable proportion of illiterate senior citizens. METHODS: Data were collected cross-sectionally by interview, using a structured questionnaire. Cognitive function was assessed using the Mini-Examen Cognoscitivo (MEC; Spanish-validated version of the Mini-Mental State Examination). RESULTS: A total of 22.4% of the subjects presented with cognitive impairment (MEC < or =22). The proportion of subjects with cognitive impairment was double in women versus men, rose to 46.2% in subjects aged > or =85 years and to 34.8% in subjects with no formal education, and was higher among those who had limitations in instrumental activities of daily living (p < 0.001). In subjects with no formal education, 25% registered mean MEC scores compatible with probable cognitive impairment. CONCLUSIONS: One of every 5 Spanish senior citizens presents with cognitive impairment, a proportion that increased among women, subjects with no formal education and with higher age.

Calidad de vida en pacientes adolescentes con epilepsia en México / Quality of life in adolescent patients with epilepsy in Mexico

Antecedentes. La epilepsia afecta todas las áreas en la vida del niño que la padece. Hay pocos estudios al respecto en poblaciones latinoamericanas. Nuestro objetivo fue evaluar la calidad de vida de los pacientes adolescentes con epilepsia con el uso del cuestionario QOLIE-31 en un hospital de tercer nivel de atención, así como identificar los factores que influyen en la misma. Pacientes y métodos. Un total de 60 pacientes adolescentes entre 12 y 17 años de la consulta externa de neurología pediátrica del Hospital Infantil “Federico Gómez” cumplió los criterios de inclusión. Se les aplicó el cuestionario QOLIE-31 entre septiembre 2008 a mayo 2009. Resultados: El 58.3% de los pacientes reportó una mala calidad de vida y solo 6.7% muy buena calidad de vida, siendo más afectados la calidad de vida en general (98.3%) y la función cognitiva (65%). Un factor muy influyente fue el antecedente de epilepsia parcial y el uso de politerapia antiepiléptica. Conclusión. La calidad de vida en los adolescentes estudiados está muy afectada por la ocurrencia de epilepsias parciales, el inicio temprano y el uso de politerapia. Se requiere un programa de atención integral con más información sobre la enfermedad...

Changes in the prevalence of overweight and obesity and their risk factors in Spain, 1987-1997.

BACKGROUND: The aim of this study was to examine the relationship of the changes in the prevalence of overweight and obesity with the changes in some of their risk factors in Spain from 1987 to 1997. METHODS: Data were obtained from three interview-based health surveys, covering representative samples of the noninstitutionalized Spanish population aged 16 years and over and undertaken in 1987 (n = 17,434), 1995 (n = 4,736), and 1997 (n = 4,678). To compensate for the different sample sizes, the 1995 and 1997 surveys were combined. Risk factors for obesity and overweight considered in this study were age, educational level, physical activity at work, physical activity during leisure time, tobacco use, alcohol consumption, and civil status. Data analysis was performed with logistic regression. RESULTS: The prevalence of overweight and obesity (body mass index >or= 25 kg/m(2)) rose from 35.6% in 1987 to 40.9% in 1995/1997 among women and from 47.1 to 56.2% among men. In the period 1987-1995/1997, the proportion of persons with secondary or university education increased by 11.3% in women and 10.8% in men. Similarly, the prevalence of intense or regular leisure-time physical activity increased by 5.7 and 6.2% in women and men, respectively. The remaining risk factors for overweight and obesity registered no substantial change in prevalence over the study period. The prevalence of overweight and obesity expected in 1995/1997, had educational level and leisure-time physical activity been the same as in 1987, is higher than the observed figures; for educational level, expected figures are 7.3 and 1.4% higher than those observed among women and men, respectively, while for leisure-time physical activity corresponding values are 2.8 and 1.6%. CONCLUSIONS: The rise in the prevalence of overweight and obesity in Spain from 1987 to 1997 may have been attenuated by an increase in leisure-time physical activity and by an improvement in educational level, particularly among women. The contribution to overweight and obesity trends attributable to physical activity at work or to tobacco and alcohol consumption appears to be small or null.