RESUMEN

BACKGROUND: For soaring birds, the ability to benefit from variable airflow dynamics is crucial, especially while crossing natural barriers such as vast water bodies during migration. Soaring birds also take advantage of warm rising air, so-called thermals, that allow birds to ascend passively to higher altitudes with reduced energy costs. Although it is well known that soaring migrants generally benefit from supportive winds and thermals, the potential of uplifts and other weather factors enabling soaring behavior remains unsolved. METHODS: In this study, we GPS-tracked 19 Red Kites, Milvus milvus, from the Central European population that crossed the Adriatic Sea on their autumn migration. Migratory tracks were annotated with weather data (wind support, side wind, temperature difference between air and surface-proxy for thermal uplift, cloud cover, and precipitation) to assess their effect on Red Kites' decisions and soaring performance along their migration across the Adriatic Sea and land. RESULTS: Wind support affected the timing of crossing over the Adriatic Sea. We found that temperature differences and horizontal winds positively affected soaring sea movement by providing lift support in otherwise weak thermals. Furthermore, we found that the soaring patterns of tracked raptors were affected by the strength and direction of prevailing winds. CONCLUSION: Thanks to modern GPS-GSM telemetry devices and available data from online databases, we explored the effect of different weather variables on the occurrence of soaring behavior and soaring patterns of migratory raptors. We revealed how wind affected the soaring pattern and that tracked birds could soar in weak thermals by utilizing horizontal winds, thus reducing energy costs of active flapping flight over vast water bodies.

RESUMEN

Carbohydrases are often incorporated into livestock feed as digestive aids to improve animal performance. AC1 is a thermostable carbohydrase with ß-1,4-glucanase, endo-cellulase, and cellobiohydrolase activity. AC1 has been expressed in corn, where it accumulates in the grain for easy inclusion in animal diets. Incorporating the enzyme in high-fiber diets (corn-soy supplemented with distiller's dry grains with solubles) that were fed to 5-week-old pigs led to a trend of decreasing viscosity of the digesta as the dose of the enzyme increased (P = 0.092). AC1 also tended to increase the apparent ileal digestibility (AID) of neutral detergent fiber (P = 0.076). When fed diets containing 2126 U/kg AC1, pigs experienced no adverse effects in terms of performance metrics (body weights, average daily gain, average daily feed intake and gain-to-feed ratio), hematology, blood chemistry or general health when compared to pigs fed a control diet that lacked AC1.

RESUMEN

The objective of this study was to determine the available P (aP) release curve for a new phytase source, GraINzyme Phytase (Agrivida Inc., Woburn, MA), which is expressed in corn containing an engineered Escherichia coli phytase called Phy02. Plant-expressed phytases are created by inserting phytase-encoding genes into plants resulting in their ability to produce seeds with increased concentrations of phytase. A total of 360 pigs (Line 200 × 400, DNA, Columbus, NE, initially 9.9 ± 0.19 kg) were used in a 21-d growth study. Pigs were weaned at approximately 21 d of age, randomly allotted to pens based on initial body weight (BW) and fed common starter diets. From days 18 to 21 postweaning, all pigs were fed a diet containing 0.11% aP. On day 21 postweaning, considered day 0 of the study, pens were blocked by BW and randomly allotted to one of eight dietary treatments with five pigs per pen and nine pens per treatment. Dietary treatments were formulated to include increasing aP derived from either an inorganic P source (0.11%, 0.19%, or 0.27% from monocalcium P) or increasing phytase (150, 250, 500, 1,000, or 1,500 FTU/kg). Diets were corn-soybean meal-based and contained 1.24% standardized ileal digestible Lys. On day 21 of the trial, one pig per pen (weighing closest to the mean pen BW) was euthanized and the right fibula was collected to determine bone ash using the nondefatted processing method. Overall (days 0 to 21), pigs fed increasing aP from inorganic P or phytase had increased (linear, P < 0.002) average daily gain (ADG), average daily feed intake (ADFI), and gain-to-feed (G:F; quadratic, P < 0.05). Bone ash weight (g) and percentage bone ash increased (linear, P < 0.001), with increasing inorganic P or added phytase. Based on the composition of the diets used in this study, the release equations developed for GraINzyme for ADG, G:F, bone ash weight, and percentage bone ash are as follows: aP = (0.255 × FTU)/(1299.969 + FTU), aP = (0.233 × FTU)/(1236.428 + FTU), aP = (45999.949 × FTU)/(462529200 + FTU), and aP = (0.272 × FTU)/(2576.581 + FTU), respectively.

RESUMEN

Antimicrobial resistance is a significant challenge for human and animal health, and developing effective antibiotic-free treatments is a strategy to help mitigate microbial resistance. The global poultry industry faces growing challenges from Eimeria-induced coccidiosis, a serious enteric disease of chickens that currently requires treatment using ionophore antibiotics. Eimeria stimulates interleukin-10 (IL-10) expression in the small intestine and caecum of infected chickens, suppressing their immune response and facilitating disease progression. Single-domain antibodies raised from llamas immunized with chicken IL-10 (cIL-10) were developed that bind cIL-10 in vitro, block cIL-10 receptor binding and induce interferon gamma (IFN-γ) secretion from cIL-10-repressed primary chicken splenocytes. Single-domain antibodies expressed in transgenic corn demonstrated significant accumulation in phenotypically normal plants. When fed to Eimeria-challenged chickens, the transgenic corn significantly improved body weight gain (equal to that of salinomycin-treated animals), normalized the feed conversion ratio (to the same level as uninfected control animals), lowered E. tenella lesion scores to those of salinomycin-treated control animals, and reduced oocyst counts below those of infected untreated control animals. Here, we propose that transgenic corn may have a role in reducing the use of antibiotics in poultry production and maintaining animal health and productivity, and may contribute to efforts against global antimicrobial resistance.

RESUMEN

A 41-d feeding trial was conducted to determine the efficacy of a corn-expressed phytase (GZ; GraINzyme, Agrivida Inc., Woburn, MA) on the live performance, bone characteristics, and P digestibility of nursery pigs fed a reduced P diet. Weaned piglets (21 ± 3 d; n = 324) were acclimated on a common diet for 7 d (phase 1) before randomization into 54 single-sex pens (5 gilt and 4 barrow pens per treatment) containing 6 pigs (6.6 ± 1.2 kg) per pen. Six treatments were fed: positive control (PC; 0.4% or 0.32% aP for phase 2 or 3 and 4, respectively), negative control (NC; 0.15% reduction in aP), and 500, 1,000, 2,000, or 4,000 FTU per kg phytase from GZ added to NC in a 3-phase feeding program. Pigs were weighed on day 1, 14, 28, and 41, and feed disappearance recorded per phase. Apparent total tract digestibility (ATTD) of P was determined by feeding chromic oxide marker (day 28 to 35) and collecting fecal samples on day 35. On day 41, 4 pigs per pen were euthanized and metacarpal bones were collected to evaluate bone breaking strength (BBS) and ash. Data were analyzed using PROC GLM of SAS (block, sex, and treatment). Treatment least squares means were separated and linear and quadratic treatment effects evaluated. Other than feed efficiency (G:F) and day 15 to 28 ADFI, the pigs fed PC were superior (P < 0.05) to NC-fed pigs in all other variables. Pigs fed ≥500 FTU per kg phytase had increased (P < 0.05) ADG and ADFI compared to NC pigs and equivalent (P > 0.05) ADG and ADFI as PC pigs from day 0 to 41. Feeding ≥500 FTU per kg phytase resulted in higher (P < 0.05) ATTD of P than both NC and PC pigs and higher (P < 0.05) BBS and bone ash weight than NC. Pigs fed 1,000 or 2,000 FTU per kg phytase had equivalent (P > 0.05) BBS and bone ash weight compared to pigs fed PC diets. Feeding 4,000 FTU per kg phytase resulted in higher (P < 0.05) day 1 to 41 ADG, ATTD of P, and bone ash weight compared to feeding ≤1,000 FTU per kg phytase or PC diets. There were linear (P < 0.05) increases in ADG, ADFI, ATTD of P, BBS, and bone ash characteristics as GZ inclusion increased. In conclusion, ≥500 FTU per kg phytase from GZ improved growth, ATTD of P, BBS, and bone ash when added to a reduced P diet and 4,000 FTU per kg phytase increased growth greater than the PC treatment.

Asunto(s)

6-Fitasa/farmacología , Alimentación Animal/análisis , Fósforo Dietético/metabolismo , Porcinos/fisiología , Animales , Huesos/fisiología , Dieta/veterinaria , Digestión/efectos de los fármacos , Heces/química , Femenino , Tracto Gastrointestinal/metabolismo , Masculino , Minerales/análisis , Proteínas de Plantas/farmacología , Distribución Aleatoria , Porcinos/crecimiento & desarrollo , Zea mays/enzimología

RESUMEN

BACKGROUND: CYP2D6 is a critical enzyme in the metabolism of tamoxifen and potentially a key determinant in breast cancer outcomes. Our study examined patients' beliefs about how the CYP2D6 genotype would affect their prognoses. METHODS: Women enrolled in a pharmacogenomic clinical trial and on tamoxifen for prevention or treatment of breast cancer underwent CYP2D6 genotyping (EM = extensive, IM = intermediate, PM = poor metabolizing alleles). The informed consent said that the purpose of the trial was to examine effects of dose adjustment based on genotype, but that clinical benefits were uncertain. Our embedded sub-study surveyed 320 patients prior to receiving their genotypes. We experimentally manipulated 6 vignettes to describe hypothetical tamoxifen treatment (no or yes) and hypothetical genotype (EM, IM or PM). For each vignette, women gave their perceived recurrence risk (RR; 0-100%). RESULTS: Women believed that genotype would not affect their RR if they did not take tamoxifen (p = 0.06). However, women believed that if prescribed tamoxifen, genotype would affect their RR (22% if EM, 30% if IM and 40% if PM, p < 0.001). CONCLUSION: Women believed that extensive tamoxifen metabolizers had better prognoses, despite study materials stating uncertainty about any benefit. The rapidly changing nature of genomic science calls for caution when communicating clinical utility.

Asunto(s)

Neoplasias de la Mama/psicología , Citocromo P-450 CYP2D6/genética , Conocimientos, Actitudes y Práctica en Salud , Recurrencia Local de Neoplasia/psicología , Educación del Paciente como Asunto/métodos , Farmacogenética , Antineoplásicos Hormonales/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Citocromo P-450 CYP2D6/metabolismo , Femenino , Genotipo , Humanos , Persona de Mediana Edad , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Pronóstico , Tamoxifeno/uso terapéutico

RESUMEN

Plant cellulosic biomass is an abundant, low-cost feedstock for producing biofuels and chemicals. Expressing cell wall-degrading (CWD) enzymes (e.g. xylanases) in plant feedstocks could reduce the amount of enzymes required for feedstock pretreatment and hydrolysis during bioprocessing to release soluble sugars. However, in planta expression of xylanases can reduce biomass yield and plant fertility. To overcome this problem, we engineered a thermostable xylanase (XynB) with a thermostable self-splicing bacterial intein to control the xylanase activity. Intein-modified XynB (iXynB) variants were selected that have <10% wild-type enzymatic activity but recover >60% enzymatic activity upon intein self-splicing at temperatures >59 °C. Greenhouse-grown xynB maize expressing XynB has shriveled seeds and low fertility, but ixynB maize had normal seeds and fertility. Processing dried ixynB maize stover by temperature-regulated xylanase activation and hydrolysis in a cocktail of commercial CWD enzymes produced >90% theoretical glucose and >63% theoretical xylose yields.

Asunto(s)

Regulación de la Temperatura Corporal/fisiología , Endo-1,4-beta Xilanasas/fisiología , Mejoramiento Genético/métodos , Inteínas/genética , Lignina/metabolismo , Plantas Modificadas Genéticamente/fisiología , Zea mays/fisiología

RESUMEN

Inteins are intervening protein domains with self-splicing ability that can be used as molecular switches to control activity of their host protein. Successfully engineering an intein into a host protein requires identifying an insertion site that permits intein insertion and splicing while allowing for proper folding of the mature protein post-splicing. By analyzing sequence and structure based properties of native intein insertion sites we have identified four features that showed significant correlation with the location of the intein insertion sites, and therefore may be useful in predicting insertion sites in other proteins that provide native-like intein function. Three of these properties, the distance to the active site and dimer interface site, the SVM score of the splice site cassette, and the sequence conservation of the site showed statistically significant correlation and strong predictive power, with area under the curve (AUC) values of 0.79, 0.76, and 0.73 respectively, while the distance to secondary structure/loop junction showed significance but with less predictive power (AUC of 0.54). In a case study of 20 insertion sites in the XynB xylanase, two features of native insertion sites showed correlation with the splice sites and demonstrated predictive value in selecting non-native splice sites. Structural modeling of intein insertions at two sites highlighted the role that the insertion site location could play on the ability of the intein to modulate activity of the host protein. These findings can be used to enrich the selection of insertion sites capable of supporting intein splicing and hosting an intein switch.

Asunto(s)

Elementos Transponibles de ADN/genética , Inteínas/genética , Modelos Moleculares , Ingeniería de Proteínas/métodos , Proteínas/química , Proteínas/genética , Área Bajo la Curva , Western Blotting , Endo-1,4-beta Xilanasas/genética , Conformación Proteica , Empalme de Proteína , Curva ROC , beta-Glucosidasa/genética

RESUMEN

In planta expression of cell wall degrading enzymes is a promising approach for developing optimized biomass feedstocks that enable low-cost cellulosic biofuels production. Transgenic plants could serve as either an enzyme source for the hydrolysis of pretreated biomass or as the primary biomass feedstock in an autohydrolysis process. In this study, two xylanase genes, Bacillus sp. NG-27 bsx and Clostridium stercorarium xynB, were expressed in maize (Zea mays) under the control of two different promoters. Severe phenotypic effects were associated with xylanase accumulation in maize, including stunted plants and sterile grains. Global expression of these xylanases from the rice ubiquitin 3 promoter (rubi3) resulted in enzyme accumulation of approximately 0.01 mg enzyme per gram dry weight, or approximately 0.1% of total soluble protein (TSP). Grain-specific expression of these enzymes from the rice glutelin 4 promoter (GluB-4) resulted in higher-level accumulation of active enzyme, with BSX and XynB accumulating up to 4.0% TSP and 16.4% TSP, respectively, in shriveled grains from selected T0 plants. These results demonstrate the potential utility of the GluB-4 promoter for biotechnological applications. The phenotypic effects of xylanase expression in maize presented here demonstrate the difficulties of hemicellulase expression in an important crop for cellulosic biofuels production. Potential alternate approaches to achieve xylanase accumulation in planta without the accompanying negative phenotypes are discussed.

Asunto(s)

Endo-1,4-beta Xilanasas/metabolismo , Regulación de la Expresión Génica de las Plantas , Glicósido Hidrolasas/metabolismo , Zea mays/metabolismo , beta-Glucosidasa/metabolismo , Bacillus/enzimología , Bacillus/genética , Clostridium/enzimología , Clostridium/genética , Grano Comestible/genética , Grano Comestible/crecimiento & desarrollo , Grano Comestible/metabolismo , Endo-1,4-beta Xilanasas/genética , Activación Enzimática , Regulación del Desarrollo de la Expresión Génica , Genes Bacterianos , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Glútenes/genética , Glútenes/metabolismo , Glicósido Hidrolasas/genética , Oryza/genética , Fenotipo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas , Semillas/genética , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Zea mays/genética , Zea mays/crecimiento & desarrollo , beta-Glucosidasa/genética

RESUMEN

The surgeon is the key "prognosis factor" for colorectal cancer. For this reason quality criteria were recently established (including minimum numbers) in order to treat patients who are entitled to the best quality of care and to improve the prognosis. The aim of this study was to critically discuss the existing demands on the surgeon based on the current literature and our own results and to formulate evidence-based quality criteria for surgical clinics. After reviewing the current literature criteria were compiled, discussed and finally presented in a summarized form. These are based on current developments on the diagnostic and therapy of large intestine and colorectal carcinoma. New developments of the German Cancer Society for planning of organ centers are incorporated. The quintessence of our study is that the number of cases alone is not decisive for the success of therapy. Important are the application of the correct surgical-oncology operation procedure, adherence to standards and the training of surgeons. Following the S3 guidelines stage-oriented therapy should additionally be carried out in a structured sequence. This includes an interdisciplinary decision making on the diagnostic and therapy strategy (tumor board). The organization structure of the hospital (teams, tumor board, emergency care with intensive care unit, emergency diagnostic and options for interventional measures) can be more important than the hospital case numbers alone. These demands which have been evaluated from published data and own results are designed to raise the therapy of colorectal cancer to the best possible level of quality and to effect a further improvement in the prognosis.

Asunto(s)

Neoplasias Colorrectales/cirugía , Garantía de la Calidad de Atención de Salud/normas , Benchmarking/normas , Competencia Clínica/normas , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Vías Clínicas/normas , Medicina Basada en la Evidencia/normas , Alemania , Adhesión a Directriz/normas , Administración Hospitalaria/normas , Humanos , Estadificación de Neoplasias , Grupo de Atención al Paciente/organización & administración , Grupo de Atención al Paciente/normas , Pronóstico , Estándares de Referencia , Tasa de Supervivencia

RESUMEN

Mammals have evolved complex regulatory systems that enable them to maintain energy homeostasis despite constant environmental challenges that limit the availability of energy inputs and their composition. Biological control relies upon intricate systems composed of multiple organs and specialized cell types that regulate energy up-take, storage, and expenditure. Because these systems simultaneously perform diverse functions and are highly integrated, they are extremely difficult to understand in terms of their individual component contributions to energy homeostasis. In order to provide improved treatments and clinical options, it is important to identify the principle genetic and molecular components, as well as the systemic features of regulation. To begin, many of these features can be discovered by integrating experimental technologies with advanced methods of analysis. This review focuses on the analysis of transcriptional data derived from microarrays and how it can complement other experimental techniques to study energy homeostasis.

RESUMEN

Metabolic engineering is a powerful methodology aimed at intelligently designing new biological pathways, systems, and ultimately phenotypes through the use of recombinant DNA technology. Built largely on the theoretical and computational analysis of chemical systems, the field has evolved to incorporate a growing number of genome scale experimental tools. This combination of rigorous analysis and quantitative molecular biology methods has endowed metabolic engineering with an effective synergism that crosses traditional disciplinary bounds. As such, there are a growing number of applications for the effective employment of metabolic engineering, ranging from the initial industrial fermentation applications to more recent medical diagnosis applications. In this review we highlight many of the contributions metabolic engineering has provided through its history, as well as give an overview of new tools and applications that promise to have a large impact on the field's future.

Asunto(s)

Biotecnología/tendencias , Metabolismo Energético/fisiología , Ingeniería de Proteínas/métodos , Ingeniería de Proteínas/tendencias , Proteínas Recombinantes/biosíntesis , Predicción

RESUMEN

BACKGROUND: Obesity is associated with insulin resistance that can often be improved by caloric restriction and weight reduction. Although many physiological changes accompanying insulin resistance and its treatment have been characterized, the genetic mechanisms linking obesity to insulin resistance are largely unknown. We used DNA microarrays and RT-PCR to investigate significant changes in hepatic gene transcription in insulin resistant, diet-induced obese (DIO)-C57/BL/6J mice and DIO-C57/BL/6J mice fasted for 48 hours, whose weights returned to baseline levels during these conditions. RESULTS: Transcriptional profiling of hepatic mRNA revealed over 1900 genes that were significantly perturbed between control, DIO, and fasting/weight reduced DIO mice. From this set, our bioinformatics analysis identified 41 genes that rigorously discriminate these groups of mice. These genes are associated with molecular pathways involved in signal transduction, and protein metabolism and secretion. Of particular interest are genes that participate in pathways responsible for modulating insulin sensitivity. DIO altered expression of genes in directions that would be anticipated to antagonize insulin sensitivity, while fasting/weight reduction partially or completely normalized their levels. Among these discriminatory genes, Sh3kbp1 and RGS3, may have special significance. Sh3kbp1, an endogenous inhibitor of PI-3-kinase, was upregulated by high-fat feeding, but normalized to control levels by fasting/weight reduction. Because insulin signaling occurs partially through PI-3-kinase, increased expression of Sh3kbp1 by DIO mice may contribute to hepatic insulin resistance via inhibition of PI-3-kinase. RGS3, a suppressor of G-protein coupled receptor generation of cAMP, was repressed by high-fat feeding, but partially normalized by fasting/weight reduction. Decreased expression of RGS3 may augment levels of cAMP and thereby contribute to increased, cAMP-induced, hepatic glucose output via phosphoenolpyruvate carboxykinase (PCK1), whose mRNA levels were also elevated. CONCLUSION: These findings demonstrate that hepatocytes respond to DIO and weight reduction by controlling gene transcription in a variety of important molecular pathways. Future studies that characterize the physiological significance of the identified genes in modulating energy homeostasis could provide a better understanding of the mechanisms linking DIO with insulin resistance.

RESUMEN

BACKGROUND AND OBJECTIVES: The compatibility of an ABO blood group independently applicable plasma, Uniplas, was explored in liver resection because patients undergoing liver resection frequently require the transfusion of plasma to compensate for blood loss and/or clotting factors. MATERIALS AND METHODS: One-hundred and twenty two patients undergoing elective liver resection were enrolled; 81 patients required plasma transfusion, while 41 did not. Of those in need of plasma, 58 were blood group A, B or AB, and 23 were blood group O. Patients were monitored up to day 7 postoperatively for signs of haemolysis and haemostasis, and viral markers were assessed at baseline and 3 weeks postoperatively. RESULTS: Uniplas transfusions of up to 50.7 ml/kg body weight were given per treatment episode, without signs of haemolysis caused by transfusion. A total of 94/99 patients (95%) were negative in the direct antiglobulin test throughout the study. Two patients, one transfused with Uniplas, the other not, had a positive direct antiglobulin test result at baseline, while three of 64 patients transfused with Uniplas demonstrated a change from having negative to intermittently positive direct antiglobulin test results without concurrent signs of haemolysis. International normalized ratio, activated partial thromboplastin time and protein C levels were maintained by transfusion of plasma (>/= 20 ml/kg body weight). No patient underwent seroconversion for human immunodeficiency virus, hepatitis B virus or hepatitis C virus. Positivity for hepatitis A virus (HAV) immunoglobulin G (IgG) in 11 patients from the Uniplas group (who tested HAV immunoglobulin M negative), together with an apparent seroconversion for parvovirus B19 seen in two patients who received Uniplas, indicated passively transferred IgG antibodies. CONCLUSIONS: No haemolysis was observed as a result of Uniplas transfusions up to 50.7 ml/kg body weight per treatment episode in patients undergoing liver resection. Moreover, transfusion (>/= 20 ml/kg body weight of Uniplas) maintained acceptable levels of international normalized ratio, activated partial thromboplastin time and protein C.

Asunto(s)

Transfusión de Componentes Sanguíneos , Hígado/cirugía , Plasma , Sistema del Grupo Sanguíneo ABO , Adulto , Anciano , Transfusión de Componentes Sanguíneos/efectos adversos , Proteínas del Sistema Complemento/análisis , Procedimientos Quirúrgicos Electivos , Femenino , Hemólisis , Hemostasis , Humanos , Relación Normalizada Internacional , Masculino , Persona de Mediana Edad , Proteína C/análisis , Triazinas

RESUMEN

We consider whether use of traceless multipole moment densities in macroscopic electromagnetism can yield physically acceptable results. For harmonic plane wave fields it is shown that a traceless electric quadrupole density yields linear constitutive relations for which the dynamical material constants (permittivity and magnetoelectric coefficients) and response fields are unphysical. We further show that, within multipole theory, these constitutive relations cannot be transformed into physically acceptable relations. Specifically, the transformed response field D is unphysical for all orders beyond the electric dipole. This contrasts with use of primitive (traced) moment densities, for which unphysical constitutive relations have been successfully transformed up to electric octopole-magnetic quadrupole order, thereby providing also the leading contribution to the ac permeability.

RESUMEN

The large number of candidate genes identified by modern high-throughput technologies require efficient methods for generating knockout phenotypes or gene silencing in order to study gene function. RNA interference (RNAi) is an efficient method that can be used for this purpose. Effective gene silencing by RNAi depends on a number of important parameters, including the dynamics of gene expression and the RNA dose. Using mouse hepatoma cells, we detail some of the principal characteristics of RNAi as a tool for gene silencing, such as the RNA dose level, RNA complex exposure time, and the time of transfection relative to gene induction, in the context of silencing a green fluorescent protein reporter gene. Our experiments demonstrate that different levels of silencing can be attained by modulating the dose level of RNA and the time of transfection and illustrate the importance of a dynamic analysis in designing robust silencing protocols. By quantifying the kinetics of RNAi-based gene silencing, we present a model that may be used to help determine key parameters in more complex silencing experiments and explore alternative gene silencing protocols.

Asunto(s)

Proteínas Luminiscentes , Interferencia de ARN , Transfección , Animales , Silenciador del Gen , Proteínas Fluorescentes Verdes , Ratones , Ratones Noqueados , Modelos Animales , Modelos Genéticos , Fenotipo

RESUMEN

The photosynthetic cyanobacterium Synechocystis sp. strain PCC 6803 uses a complex genetic program to control its physiological response to alternating light conditions. To study this regulatory program time-series experiments were conducted by exposing Synechocystis sp. to serial perturbations in light intensity. In each experiment whole-genome DNA microarrays were used to monitor gene transcription in 20-min intervals over 8- and 16-h periods. The data was analyzed using time-lagged correlation analysis, which identifies genetic interaction networks by constructing correlations between time-shifted transcription profiles with different levels of statistical confidence. These networks allow inference of putative cause-effect relationships among the organism's genes. Using light intensity as our initial input signal, we identified six groups of genes whose time-lagged profiles possessed significant correlation, or anti-correlation, with the light intensity. We expanded this network by using the average profile from each group of genes as a seed, and searching for other genes whose time-lagged profiles possessed significant correlation, or anti-correlation, with the group's average profile. The final network comprised 50 different groups containing 259 genes. Several of these gene groups possess known light-stimulated gene clusters, such as Synechocystis sp. photosystems I and II and carbon dioxide fixation pathways, while others represent novel findings in this work.

Asunto(s)

Cianobacterias/genética , Perfilación de la Expresión Génica , Genes Bacterianos , Genoma Bacteriano , Regulación Bacteriana de la Expresión Génica , Luz , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Factores de Tiempo

RESUMEN

An important objective in postgenomic biology is to link gene expression to function by developing physiological networks that include data from the genomic and functional levels. Here, we develop a model for the analysis of time-dependent changes in metabolites, fluxes, and gene expression in a hepatic model system. The experimental framework chosen was modulation of extracellular glutamine in confluent cultures of mouse Hepa1-6 cells. The importance of glutamine has been demonstrated previously in mammalian cell culture by precipitating metabolic shifts with glutamine depletion and repletion. Our protocol removed glutamine from the medium for 24 h and returned it for a second 24 h. Flux assays of glycolysis, the tricarboxylic acid (TCA) cycle, and lipogenesis were used at specified intervals. All of these fluxes declined in the absence of glutamine and were restored when glutamine was repleted. Isotopomer spectral analysis identified glucose and glutamine as equal sources of lipogenic carbon. Metabolite measurements of organic acids and amino acids indicated that most metabolites changed in parallel with the fluxes. Experiments with actinomycin D indicated that de novo mRNA synthesis was required for observed flux changes during the depletion/repletion of glutamine. Analysis of gene expression data from DNA microarrays revealed that many more genes were anticorrelated with the glycolytic flux and glutamine level than were correlated with these indicators. In conclusion, this model may be useful as a prototype physiological regulatory network where gene expression profiles are analyzed in concert with changes in cell function.

Asunto(s)

Glutamina/farmacología , Neoplasias Hepáticas Experimentales/metabolismo , Modelos Biológicos , Transcripción Genética , Animales , Línea Celular Tumoral , Ciclo del Ácido Cítrico , Perfilación de la Expresión Génica , Glutamina/fisiología , Glucólisis , Cinética , Metabolismo de los Lípidos , Neoplasias Hepáticas Experimentales/genética , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos

RESUMEN

AIM: The standard treatment for patients with clinically resectable rectal cancer is surgery. Postoperative radiochemotherapy (RCT) is recommended for advanced disease (pT3/4 or pN+). In recent years, encouraging results of pre-operative radiotherapy have been reported. This prospective randomized phase-III-trial (CAO/ARO/AIO-94) compares the efficacy of neoadjuvant RCT to standard postoperative RCT. We report on the design of the study and first results with regard to toxicity of RCT and postoperative morbidity. PATIENTS AND METHODS: Patients with locally advanced operable rectal cancer (uT3/4 or uN+, Mason CS III/IV) were randomly assigned to pre or postoperative RCT: A total dose of 50.4 Gy (single dose 1.8 Gy) was applied to the tumour and the pelvic lymph nodes. 5-FU (1000 mg/m2/d) was administered concomitantly in the 1th and 5th week of radiation as 120 h-continuous infusion. Four additional cycles of 5-FU-chemotherapy (500 mg/m2/d, i.v.-bolus) were applied. RCT was identical in both arms except for a small-volume boost of 5.4 Gy postoperatively. The time interval between RCT and surgery was 4-6 weeks in both arms. Techniques of surgery were standardized and included total mesorectal excision. Primary endpoints of the study are 5-year survival and local and distant control. Secondary endpoints include the rate of curative (R0) resection and sphincter saving procedures, toxicity of RCT, surgical complications and quality of life. RESULTS: As of July 2002, 805 patients were randomized from 26 participating institutions. Acute toxicity (WHO) of RCT was low, with less than 15% of patients experiencing grade 3 or higher toxicity: The principal toxicity was diarrhea, with 12% in the postoperative RCT-arm and 11% in the pre-operative RCT-arm having grade 3-, and 1% in either arm having grade 4-diarrhea. Erythema, nausea and leukopenia were the next common toxicities, with less than 3% of patients in either arm suffering grade 3 or greater leukopenia or nausea. Postoperative complication rates were similar in both arms, with 12% (postop. RCT) and 12% (pre-op. RCT) of patients, respectively, suffering from anastomotic leakage, 3% (postop. RCT) and 3% (pre-op. RCT) from postoperative bleeding, and 6% (postop. RCT) and 4% (pre-op. RCT) from delayed wound healing. CONCLUSION: The patient accrual to the trial is satisfactory. Neoadjuvant RCT is well tolerated and bears no higher risk for postoperative morbidity.

Asunto(s)

Antimetabolitos Antineoplásicos/administración & dosificación , Fluorouracilo/administración & dosificación , Terapia Neoadyuvante , Neoplasias del Recto/tratamiento farmacológico , Neoplasias del Recto/radioterapia , Adulto , Anciano , Antimetabolitos Antineoplásicos/efectos adversos , Quimioterapia Adyuvante/efectos adversos , Femenino , Fluorouracilo/efectos adversos , Alemania , Humanos , Masculino , Persona de Mediana Edad , Terapia Neoadyuvante/efectos adversos , Estadificación de Neoplasias , Selección de Paciente , Complicaciones Posoperatorias/etiología , Calidad de Vida , Dosificación Radioterapéutica , Radioterapia Adyuvante/efectos adversos , Neoplasias del Recto/patología , Neoplasias del Recto/cirugía , Análisis de Supervivencia , Resultado del Tratamiento

RESUMEN

Taking KDP as an example of a uniaxial crystal, we analyze contributions to its quadratic electro-optic response with a view to explaining differences in published values of certain quadratic coefficients. In an eigenvalue theory of light propagation we show that the linear electro-optic coefficient contributes to the quadratic response, even under ideal laboratory conditions. In addition, the effect of imperfect crystal cutting and alignment is investigated by means of computer calculations based on the Jones calculus. It is found that, for relatively small inaccuracies, the calculated values of the quadratic coefficient g(xyxy) are approximately two orders of magnitude greater when measured with a static field than with a dynamic one. This finding could explain the observed spread in some results for KDP-type crystals.