Assessing and harnessing updated polyketide synthase modules through combinatorial engineering.

The modular nature of polyketide assembly lines and the significance of their products make them prime targets for combinatorial engineering. The recently updated module boundary has been successful for engineering short synthases, yet larger synthases constructed using the updated boundary have not been investigated. Here we describe our design and implementation of a BioBricks-like platform to rapidly construct 5 triketide, 25 tetraketide, and 125 pentaketide synthases to test every module combination of the pikromycin synthase. Anticipated products are detected from 60% of the triketide synthases, 32% of the tetraketide synthases, and 6.4% of the pentaketide synthases. We determine ketosynthase gatekeeping and module-skipping are the principal impediments to obtaining functional synthases. The platform is also employed to construct active hybrid synthases by incorporating modules from the erythromycin, spinosyn, and rapamycin assembly lines. The relaxed gatekeeping of a ketosynthase in the rapamycin synthase is especially encouraging in the quest to produce designer polyketides.

Quantitative morphological analysis of Deinococcus radiodurans elucidates complex dose-dependent nucleoid condensation during recovery from ionizing radiation.

The extremophile Deinococcus radiodurans maintains a highly organized and condensed nucleoid as its default state, possibly contributing to its high tolerance to ionizing radiation (IR). Previous studies of the D. radiodurans nucleoid were limited by reliance on manual image annotation and qualitative metrics. Here, we introduce a high-throughput approach to quantify the geometric properties of cells and nucleoids using confocal microscopy, digital reconstructions of cells, and computational modeling. We utilize this novel approach to investigate the dynamic process of nucleoid condensation in response to IR stress. Our quantitative analysis reveals that at the population level, exposure to IR induced nucleoid compaction and decreased the size of D. radiodurans cells. Morphological analysis and clustering identified six distinct sub-populations across all tested experimental conditions. Results indicate that exposure to IR induced fractional redistributions of cells across sub-populations to exhibit morphologies associated with greater nucleoid condensation and decreased the abundance of sub-populations associated with cell division. Nucleoid-associated proteins (NAPs) may link nucleoid compaction and stress tolerance, but their roles in regulating compaction in D. radiodurans are unknown. Imaging of genomic mutants of known and suspected NAPs that contribute to nucleoid condensation found that deletion of nucleic acid-binding proteins, not previously described as NAPs, can remodel the nucleoid by driving condensation or decondensation in the absence of stress and that IR increased the abundance of these morphological states. Thus, our integrated analysis introduces a new methodology for studying environmental influences on bacterial nucleoids and provides an opportunity to further investigate potential regulators of nucleoid condensation.IMPORTANCEDeinococcus radiodurans, an extremophile known for its stress tolerance, constitutively maintains a highly condensed nucleoid. Qualitative studies have described nucleoid behavior under a variety of conditions. However, a lack of quantitative data regarding nucleoid organization and dynamics has limited our understanding of the regulatory mechanisms controlling nucleoid organization in D. radiodurans. Here, we introduce a quantitative approach that enables high-throughput quantitative measurements of subcellular spatial characteristics in bacterial cells. Applying this to wild-type or single-protein-deficient populations of D. radiodurans subjected to ionizing radiation, we identified significant stress-responsive changes in cell shape, nucleoid organization, and morphology. These findings highlight this methodology's adaptability and capacity for quantitatively analyzing the cellular response to stressors for screening cellular proteins involved in bacterial nucleoid organization.

Assessing and harnessing updated polyketide synthase modules through combinatorial engineering.

The modular nature of polyketide assembly lines and the significance of their products make them prime targets for combinatorial engineering. While short synthases constructed using the recently updated module boundary have been shown to outperform those using the traditional boundary, larger synthases constructed using the updated boundary have not been investigated. Here we describe our design and implementation of a BioBricks-like platform to rapidly construct 5 triketide, 25 tetraketide, and 125 pentaketide synthases from the updated modules of the Pikromycin synthase. Every combinatorial possibility of modules 2-6 inserted between the first and last modules of the native synthase was constructed and assayed. Anticipated products were observed from 60% of the triketide synthases, 32% of the tetraketide synthases, and 6.4% of the pentaketide synthases. Ketosynthase gatekeeping and module-skipping were determined to be the principal impediments to obtaining functional synthases. The platform was also used to create functional hybrid synthases through the incorporation of modules from the Erythromycin, Spinosyn, and Rapamycin assembly lines. The relaxed gatekeeping observed from a ketosynthase in the Rapamycin synthase is especially encouraging in the quest to produce designer polyketides.

Análisis de la composición corporal empleando parámetros bioeléctricos en la población deportiva cubana / Body composition analysis using bioelectrical parameters in Cuban sporting population

Objetivos: La propuesta de este estudio es proveer datos de parámetros bioeléctricos de la población deportiva cubana, particularmente: resistencia (R), reactancia (Xc), impedancia (Z), ángulo de fase (AF), y los componentes del vector impedancia de acuerdo al análisis del vector bioeléctrico (BIVA), estableciendo valores de referencia en atletas de alto rendimiento. Material y método: Se les realizó el análisis de bioimpedancia eléctrica (BIA) a 943 deportistas cubanos (620 masculinos, 323 femeninos) de 26 deportes diferentes. Los parámetros bioeléctricos R, Xc, Z y PA fueron obtenidos a una frecuencia de 50-kHz y en la gama de 1 a 1.000 kHz usando un analizador multifrecuencia. De estos parámetros, fueron calculados cinco índices (R/Estatura, Xc/ Estatura, Z/Estatura, reactancia en paralelo, y resistencia en paralelo) a 50 kHz. Se obtuvo el análisis del vector de bioimpedancia eléctrica (BIVA) y se realizó un modelado Cole-Cole. Una vez estimado R/Estatura y Xc/Estatura, fue realizado el análisis de BIVA. La media y la desviación estándar fueron calculadas para todas las variables. La prueba t fue usada para detectar las diferencias entre ambos sexos.Los BIVA fueron comparados usando la T2 de Hotelling. Para contrastar la hipótesis nula de igualdad entre los parámetros examinados, se empleó el nivel de significación de p <0,05. Resultados: En comparación a la población masculina, la población femenina tuvo mayor R, R/Estatura, Xc, Xc/Estatura, Z, Z/Estatura y menor PA (p<0,05). La precisión de BIVA fue diferente entre los sexos (p <0,05) y de acuerdo al índice de masa corporal (≤25 y >25). Las diferencias entre deportes estuvieron relacionadas al valor de Z y AF. Conclusiones: El estudio mostró que la variabilidad de R, Xc, Z y AF dependió del género, de las características de la masa corporal de la población estudiada, y del deporte

Objectives: The purpose of this study is to provide data on bioelectrical parameters in Cuban sport population, particularly: resistance (R), reactance (Xc), impedance (Z), impedance vector component according to bioelectrical impedance vector analysis (BIVA) and phase angle (PhA), establishing references values on athletes of high performance level. Material and method: We performed bioelectrical impedance analysis (BIA) in 943 Cuban athletes (620 male, 323 female) of 26 sports modalities. Bioelectric parameters R, Xc, Z and PhA were obtained at 50-kHz frequency and multi-frequency from 1 to 1000 kHz using a multi-frequency measuring device. From these parameters, five bioimpedance ratios were calculated (R/Height, Xc/ Height, Z/ Height, the reactance paralate, and resistence paralate) at 50 kHz. Bioelectrical impedance vectors analysis (BIVA) and Cole-Cole modeled were performed. Once R/Height and Xc/Height were estimated, the BIVA was performed. Mean and standard deviations were calculated for all variables. Unpaired t-test was used to detect differences between sexes. BIVA vectors were compared using Hotelling’s T2 test. To eliminate a null hypothesis about the equality of the examined parameters, we used the level of probability of p<0.05. Results: Compared to male population, female population had higher R, R/ Height, Xc, Xc/ Height, Z, Z/ Height and lower PhA. The accuracy of specific BIVA was different in the two sexes (p<0.05) and according to the Body Mass Index (≤25 and >25). Differences between sports were found in order to Z and PhA. Conclusions: The study showed that variability of R, Xc, Z and PhA measures depends on gender, body mass characteristics of the study population, and sport