ABSTRACT
In our recent article "In vivo evolution of metabolic pathways by homeologous recombination in mitotic cells" we proposed a useful alternative to directed evolution methods that permits the generation of yeast cell libraries containing recombinant metabolic pathways from counterpart genes. The methodology was applied to generate single mosaic genes and intragenic mosaic pathways. We used flavonoid metabolism genes as a working model to assembly and express evolved pathways in DNA repair deficient cells. The present commentary revises the principles of gene and pathway mosaicism and explores the scope and perspectives of our results as an additional tool for synthetic biology.
Subject(s)
Directed Molecular Evolution/methods , Homologous Recombination , Metabolic Engineering/methods , Mitosis/genetics , Saccharomyces cerevisiaeABSTRACT
We describe a rapid and highly efficient method for the assembly, recombination, targeted chromosomal integration and regulatable expression of mosaic metabolic pathways by homeologous recombination in DNA repair deficient yeast cells. We have assembled and recombined 23kb pathways containing all the genes encoding enzymes for the production of flavonoids, a group of plant secondary metabolites of nutritional and agricultural value. The mosaic genes of the pathways resulted from pair-wise recombination of two nonidentical (homeologous) wild-type genes. The recombination events occurred simultaneously in the cell. Correctly assembled mosaic gene clusters could only be observed in DNA repair deficient strains. Thus, libraries of intragenic mosaic pathways were generated. Randomly isolated clones were screened for their ability to produce flavonoids such as kaempferol, phloretin and galangin. Thus, the functionality of the recombinant pathways was proven. Additionally, significant higher concentrations of metabolites such as naringenin, pinocembrin and dihydrokaempferol were detected. Further analysis also revealed the production of different aromatic compounds such as styrene, hydroxystyrene, phloretic acid and other molecules. We show that the in vivo homeologous recombination strategy can generates libraries of intragenic mosaic pathways producing a high diversity of phenylpropanoid compounds.
Subject(s)
Directed Molecular Evolution/methods , Homologous Recombination , Metabolic Engineering/methods , Mitosis/genetics , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolismABSTRACT
Membrane electropermeabilization relies on the transient permeabilization of the plasma membrane of cells submitted to electric pulses. This method is widely used in cell biology and medicine due to its efficiency to transfer molecules while limiting loss of cell viability. However, very little is known about the consequences of membrane electropermeabilization at the molecular and cellular levels. Progress in the knowledge of the involved mechanisms is a biophysical challenge. As a transient loss of membrane cohesion is associated with membrane permeabilization, our main objective was to detect and visualize at the single-cell level the incidence of phospholipid scrambling and changes in membrane order. We performed studies using fluorescence microscopy with C6-NBD-PC and FM1-43 to monitor phospholipid scrambling and membrane order of mammalian cells. Millisecond permeabilizing pulses induced membrane disorganization by increasing the translocation of phosphatidylcholines according to an ATP-independent process. The pulses induced the formation of long-lived permeant structures that were present during membrane resealing, but were not associated with phosphatidylcholine internalization. These pulses resulted in a rapid phospholipid flip/flop within less than 1s and were exclusively restricted to the regions of the permeabilized membrane. Under such electrical conditions, phosphatidylserine externalization was not detected. Moreover, this electrically-mediated membrane disorganization was not correlated with loss of cell viability. Our results could support the existence of direct interactions between the movement of membrane zwitterionic phospholipids and the electric field.
Subject(s)
Cell Membrane/metabolism , Phospholipids/metabolism , Adenosine Triphosphate/metabolism , Animals , CHO Cells , Cell Line , Cell Membrane Permeability , Cell Survival/physiology , Cricetulus , Electroporation/methods , Phosphatidylcholines/metabolismABSTRACT
Antecedentes: El abordaje laparoscopico en la restitución del tránsito luego de operación tipo Hartmann es una opción razonable, aunque con alto índice de conversión. Objetivo: Análisis de resultados en la restitución de Hartmann por laparoscopia y convencional. Diseño: Estudio retrospectivo comparativo. Población: Pacientes operados en un período (1991/2009) en forma sucesiva. Abordaje laparoscópico, 29; convencional, 30. Método: Evaluación del dolor, recuperación del tránsito intestinal, internación y recuperación laboral. La conversión fue analizada en relación a la curva de aprendizaje, antecedente de peritonitis y tiempo transcurrido desde la primer intervención. Resultados: Causas principales de la primera cirugía: Enfermedad diverticular, 27 y cáncer, 15. Hubo 6 accidentes intraoperatorios, laparoscópico, 2; convencional, 4 (Lesión del delgado) (p=0,42%). Las complicaciones fueron 3, y 5 respectivamente. (p= 0,67).La mortalidad fue de 2 para el grupo laparoscópico. (p=0,49).Hubo 5 conversiones (17,2 %). Sin diferencias entre tiempo transcurrido de la primer cirugía (p = 0,32); curva de aprendizaje (p = 0,16) y antecedente de peritonitis (0,65), tiempo de internación (p=0,80), recuperación laboral (p=0,87), curva de aprendizaje (p=0,16), tiempo transcurrido de la primer cirugía (p=0,32) y presencia de peritonitis (p=0,65). El dolor y la recuperación del tránsito fueron menores en el grupo laparoscópico (p=0.0001). Conclusión: La restitución del tránsito luego de operación de Hartmann mediante laparoscopia es posible; aunque con un alto índice de conversión en relación al resto de la cirugía colorrectal, con una morbimortalidad aceptable, menor dolor y recuperación del tránsito intestinal; con respecto a la técnica abierta.
Subject(s)
Male , Female , Adult , Aged , Colon , Laparoscopy , Neoplasms , Colostomy , General SurgeryABSTRACT
Antecedentes: El abordaje laparoscopico en la restitución del tránsito luego de operación tipo Hartmann es una opción razonable, aunque con alto índice de conversión. Objetivo: Análisis de resultados en la restitución de Hartmann por laparoscopia y convencional. Diseño: Estudio retrospectivo comparativo. Población: Pacientes operados en un período (1991/2009) en forma sucesiva. Abordaje laparoscópico, 29; convencional, 30. Método: Evaluación del dolor, recuperación del tránsito intestinal, internación y recuperación laboral. La conversión fue analizada en relación a la curva de aprendizaje, antecedente de peritonitis y tiempo transcurrido desde la primer intervención. Resultados: Causas principales de la primera cirugía: Enfermedad diverticular, 27 y cáncer, 15. Hubo 6 accidentes intraoperatorios, laparoscópico, 2; convencional, 4 (Lesión del delgado) (p=0,42%). Las complicaciones fueron 3, y 5 respectivamente. (p= 0,67). La mortalidad fue de 2 para el grupo laparoscópico. (p=0,49). Hubo 5 conversiones (17,2 %). Sin diferencias entre tiempo transcurrido de la primer cirugía (p = 0,32); curva de aprendizaje (p = 0,16) y antecedente de peritonitis (0,65), tiempo de internación (p=0,80), recuperación laboral (p=0,87), curva de aprendizaje (p=0,16), tiempo transcurrido de la primer cirugía (p=0,32) y presencia de peritonitis (p=0,65). El dolor y la recuperación del tránsito fueron menores en el grupo laparoscópico (p=0.0001). Conclusión: La restitución del tránsito luego de operación de Hartmann mediante laparoscopia es posible; aunque con un alto índice de conversión en relación al resto de la cirugía colorrectal, con una morbimortalidad aceptable, menor dolor y recuperación del tránsito intestinal; con respecto a la técnica abierta.(AU)
Background: The laparoscopic approach in the restitution of the intestinal transit after operation type Hartmann is a reasonable option, though with high index of conversion. Objetive: Analysis of results in Hartmanns restitution by laparoscopic and conventional approach. Design: Retrospective, comparative study. Population: Patients produced in a period (1991/2009) in successive form. Laparoscopic approach 29; conventional, 30. Method: Evaluation of the pain, recovery of the intestinal movement, surgery and labor recovery. The conversion went in relation to the curve of learning, precedent of peritonitis and time passed from the first intervention. Results: Mainsprings of first surgery: Disease diverticular, 27 and cancer, 15. There were 6 intraoperative accidents, laparoscópic, 2; conventional, 4 ( Intestinal Injury) (p=0,42 %). The complications were 3, and 5 respectively. (P = 0,67) The mortality was of 2 patients for the laparoscopic group. (P=0,49) There were 5 conversions (17.2 %). Without differences between time passed of the first surgery (p = 0,32); curve of learning (p = 0,16) and precedent of peritonitis (0,65), time of recovery (p=0,80), labor recovery (p=0,87), curved of learning (p=0,16), time passed of the first surgery (p=0,32) and presence of peritonitis (p=0,65). The pain and the recovery of the intestinal movements were minor in the laparoscopic group (p=0.0001) Conclusions: The restitution of the intestinal transit after Hartmanns operation by laparoscopic approach is possible; though with a high index of conversion in relation to the rest of the colorectal surgery, with an acceptable complication and mortality rate, minor pain and recovery of the intestinal transit; that the open approach.(AU)
ABSTRACT
Membrane permeabilization is achieved via numerous techniques involving the use of molecular agents such as peptides used in antimicrobial therapy. Although high efficiency is reached, the permeabilization mechanism remains global with a noticeable lack of control. To achieve localized control and more gradual increase in membrane perturbation, we have developed hydrophobically modified poly(acrylic acid) amphiphilic copolymers with light-responsive azobenzene hydrophobic moieties. We present evidence for light triggered membrane permeabilization in the presence azobenzene-modified polymers (AMPs). Exposure to UV or blue light reversibly switches the polarity of the azobenzene (cis-trans isomerization) in AMPs, hence controlling AMP-loaded lipid vesicles permeabilization via in situ activation. Release of encapsulated probes was studied by microscopy on isolated AMP-loaded giant unilamellar vesicles (pol-GUVs). We show that in pH and ionic strength conditions that are biologically relevant pol-GUVs are kept impermeable when they contain predominantly cis-AMPs but become leaky with no membrane breakage upon exposure to blue light due to AMPs switch to a trans-apolar state. In addition, we show that AMPs induce destabilization of plasma membranes when added to mammal cells in their trans-apolar state, with no loss of cell viability. These features make AMPs promising tools for remote control of cell membrane permeabilization in mild conditions.
Subject(s)
Acrylic Resins/chemistry , Cell Membrane Permeability , Light , Membrane Lipids/chemistry , Acrylic Resins/chemical synthesis , Animals , Azo Compounds/chemistry , COS Cells , Cell Survival , Cells, Cultured , Chlorocebus aethiops , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Molecular Structure , Osmolar Concentration , Surface PropertiesABSTRACT
Drug molecules must cross multiple cell membrane barriers to reach their site of action. We present evidence that one of the largest classes of pharmaceutical drug molecules, the cationic amphiphilic drugs (CADs), does so via a catalytic reaction that degrades the phospholipid fabric of the membrane. We find that CADs partition rapidly to the polar-apolar region of the membrane. At physiological pH, the protonated groups on the CAD catalyse the acid hydrolysis of the ester linkage present in the phospholipid chains, producing a fatty acid and a single-chain lipid. The single-chain lipids rapidly destabilize the membrane, causing membranous fragments to separate and diffuse away from the host. These membrane fragments carry the drug molecules with them. The entire process, from drug adsorption to drug release within micelles, occurs on a time-scale of seconds, compatible with in vivo drug diffusion rates. Given the rate at which the reaction occurs, it is probable that this process is a significant mechanism for drug transport.
Subject(s)
Lipid Bilayers/metabolism , Surface-Active Agents/pharmacokinetics , Biological Transport, Active , Cations , Diffusion , Drug Delivery Systems , In Vitro Techniques , Lipid Bilayers/chemistry , Liquid Crystals , Magnetic Resonance Spectroscopy , Microscopy, Fluorescence , Models, Biological , Phospholipids/chemistry , Phospholipids/metabolism , Scattering, Radiation , Surface-Active Agents/chemistry , X-RaysABSTRACT
El cáncer del conducto anal es relativamente raro, constituye el 1 a 2 por ciento de los tumores gastrointestinales y el 3 a 4 por ciento de los colorrectoanales. El adenocarcinoma representa entre el 5 y 17 por ciento de los cánceres anales. El objetivo del trabajo es una presentación clínica de un paciente operado y la actualización bibliográfica en los aspectos anatomopatológicos de clasificación, origen, diagnóstico y tratamiento(AU)
Subject(s)
Male , Humans , Rectal Neoplasms/etiology , Rectal Neoplasms/surgery , Adenocarcinoma/surgery , Adenocarcinoma/therapy , Rectal Fistula/etiology , Fournier Gangrene/surgery , Fournier Gangrene/therapy , Crohn Disease/diagnosis , Crohn Disease/surgeryABSTRACT
El cáncer del conducto anal es relativamente raro, constituye el 1 a 2 por ciento de los tumores gastrointestinales y el 3 a 4 por ciento de los colorrectoanales. El adenocarcinoma representa entre el 5 y 17 por ciento de los cánceres anales. El objetivo del trabajo es una presentación clínica de un paciente operado y la actualización bibliográfica en los aspectos anatomopatológicos de clasificación, origen, diagnóstico y tratamiento
Subject(s)
Male , Humans , Adenocarcinoma , Crohn Disease , Rectal Fistula/etiology , Fournier Gangrene , Rectal Neoplasms/surgery , Rectal Neoplasms/etiologyABSTRACT
Background: Pressure ulcers are a common complication among elderly patients confined to bed for long periods. The Braden scale is a commonly used risk assessment tool. Aim: To evaluate the use of Braden scale. Patients and methods: Seventy women aged 61 to 96 years, admitted to the Internal Medicine Service of Barros Luco-Trudeau Hospital, were studied. Their risk was evaluated using the Braden scale. The presence of pressure ulcer was diagnosed according to the National Pressure Ulcer Advisory Panel on admition, two weeks later and at discharge. Results: On admission, mean Braden scale score was 16.6±2.8 and 34 women had a score of 16 or less, that is considered of risk. Twenty five women (20 with a score of 16 or less) developed pressure ulcers, mostly superficial. The odds ratio of a score of 16 or less for the development of ulcers was 4.2 (95 percent CI 1.8-11.7, p <0.001). The sensitivity and specificity of such score were 80 and 69 percent respectively. Conclusions: The Braden scale predicts the risk of developing pressure ulcers with a good sensitivity and specificity in female elderly patients (Rev MÚd Chile 2004; 132: 595-600).
Subject(s)
Humans , Male , Female , Aged , Predictive Value of Tests , Pressure Ulcer/diagnosis , Pressure Ulcer/epidemiologyABSTRACT
Objetivo. Aplicar y validar en una población chilena el "Indice de Función Sexual Femenina" establecido en el International Consensus Development Conference on Female Sexual Dysfunctions. Material y método. 383 mujeres sanas de 20 a 59 a¤os con actividad sexual, beneficiarias del Centro de Salud "Carol Urzúa". Instrumento: cuestionario de 19 preguntas, agrupadas en seis dominios: deseo, excitación, lubricación, orgasmo, satisfacción y dolor. Análisis estadístico: Se utilizó ANOVA, Kruskall-Wallis, Chi cuadrado, regresión logística y alpha de Cronbach. Resultados. Edad media: 35,3ñ10,9 a¤os, casadas (50,4 por ciento) o conviviente (17,0 por ciento), con educación media (48,2 por ciento). La consistencia interna del test fue buena (>0,70). La sexualidad logra su máxima expresión a los 35-40 a¤os (puntaje: 29,1ñ4,9) para caer posteriormente (21,0ñ6,0), especialmente el deseo y excitación. Después de los 44 a¤os se incrementa el riesgo de disfunción sexual (OR:3,6; IC: 2,1-6,3; p< 0,0001). La mayor educación y la estabilidad de pareja disminuyen el riesgo (OR: 0,45; IC:0,28-0,80; p< 0,005 y OR:0,58; IC:0,35-0,98; p< 0,05 respectivamente). Conclusiones. El Indice de Función Sexual Femenino es un instrumento sencillo de aplicar, con propiedades psicométricas adecuadas que permite evaluar la sexualidad en diferentes etapas de la vida. Es adecuado para estudios epidemiológicos y clínicos.
Subject(s)
Humans , Female , Sexual Behavior/statistics & numerical data , Sexual Behavior/psychology , Sexual Dysfunctions, Psychological/epidemiology , SexualityABSTRACT
The loss of sexuality observed in the climacteric period is difficult to evaluate. An important advance has been the development of the Female Sexual Function Index (FSFI), a test based on the norms of the International Consensus Development Conference on Sexual Female Dysfunctions. Aim: To study the effects of hormone replacement therapy (HRT) on sexuality, applying the FSFI. Material and methods: The FSFI was applied to 300 healthy women between 45-64 years, sexually active, beneficiaries of the Southern Metropolitan Health Service. Results: The mean age of the sample studied was 51±5 years, 27% were HRT users, 21% had had an hysterectomy and 98% had a stable couple. The total score of the FSFI decreased from 27.3±5.8 in women between 45 and 49 years of age to 19.3±7.0 in women between 60 and 64 years (p <0.01). A significantly better sexuality was observed in HRT users, with FSFI scores of 28.1±5.5 and 24.6±6.8 in HRT users and non users, respectively (p <0.01). Women on HRT obtained a higher score in all of the test domains, especially in lubrication, orgasm and sexual satisfaction. Conclusions: Female sexuality decreases with aging. HRT users have a better sexual function than non users (Rev Méd Chile 2003; 131: 1251-55 ).
