A versatile platform strain for high-fidelity multiplex genome editing.

Precision genome editing accelerates the discovery of the genetic determinants of phenotype and the engineering of novel behaviors in organisms. Advances in DNA synthesis and recombineering have enabled high-throughput engineering of genetic circuits and biosynthetic pathways via directed mutagenesis of bacterial chromosomes. However, the highest recombination efficiencies have to date been reported in persistent mutator strains, which suffer from reduced genomic fidelity. The absence of inducible transcriptional regulators in these strains also prevents concurrent control of genome engineering tools and engineered functions. Here, we introduce a new recombineering platform strain, BioDesignER, which incorporates (i) a refactored λ-Red recombination system that reduces toxicity and accelerates multi-cycle recombination, (ii) genetic modifications that boost recombination efficiency, and (iii) four independent inducible regulators to control engineered functions. These modifications resulted in single-cycle recombineering efficiencies of up to 25% with a 7-fold increase in recombineering fidelity compared to the widely used recombineering strain EcNR2. To facilitate genome engineering in BioDesignER, we have curated eight context--neutral genomic loci, termed Safe Sites, for stable gene expression and consistent recombination efficiency. BioDesignER is a platform to develop and optimize engineered cellular functions and can serve as a model to implement comparable recombination and regulatory systems in other bacteria.

The three-dimensional architecture of a bacterial genome and its alteration by genetic perturbation.

We have determined the three-dimensional (3D) architecture of the Caulobacter crescentus genome by combining genome-wide chromatin interaction detection, live-cell imaging, and computational modeling. Using chromosome conformation capture carbon copy (5C), we derive ~13 kb resolution 3D models of the Caulobacter genome. The resulting models illustrate that the genome is ellipsoidal with periodically arranged arms. The parS sites, a pair of short contiguous sequence elements known to be involved in chromosome segregation, are positioned at one pole, where they anchor the chromosome to the cell and contribute to the formation of a compact chromatin conformation. Repositioning these elements resulted in rotations of the chromosome that changed the subcellular positions of most genes. Such rotations did not lead to large-scale changes in gene expression, indicating that genome folding does not strongly affect gene regulation. Collectively, our data suggest that genome folding is globally dictated by the parS sites and chromosome segregation.

Caulobacter chromosome in vivo configuration matches model predictions for a supercoiled polymer in a cell-like confinement.

We measured the distance between fluorescent-labeled DNA loci of various interloci contour lengths in Caulobacter crescentus swarmer cells to determine the in vivo configuration of the chromosome. For DNA segments less than about 300 kb, the mean interloci distances, , scale as n(0.22), where n is the contour length, and cell-to-cell distribution of the interloci distance r is a universal function of r/n(0.22) with broad cell-to-cell variability. For DNA segments greater than about 300 kb, the mean interloci distances scale as n, in agreement with previous observations. The 0.22 value of the scaling exponent for short DNA segments is consistent with theoretical predictions for a branched DNA polymer structure. Predictions from Brownian dynamics simulations of the packing of supercoiled DNA polymers in an elongated cell-like confinement are also consistent with a branched DNA structure, and simulated interloci distance distributions predict that confinement leads to "freezing" of the supercoiled configuration. Lateral positions of labeled loci at comparable positions along the length of the cell are strongly correlated when the longitudinal locus positions differ by <0.16 µm. We conclude that the chromosome structure is supercoiled locally and elongated at large length scales and that substantial cell-to-cell variability in the interloci distances indicates that in vivo crowding prevents the chromosome from reaching an equilibrium arrangement. We suggest that the force causing rapid transport of loci remote from the parS centromere to the distal cell pole may arise from the release at the polar region of potential energy within the supercoiled DNA.

High-throughput DNA engineering by mating bacteria.

To reduce the operational friction and scale DNA engineering, we report here an in vivo DNA assembly technology platform called SCRIVENER (Sequential Conjugation and Recombination for In Vivo Elongation of Nucleotides with low ERrors). SCRIVENER combines bacterial conjugation, in vivo DNA cutting, and in vivo homologous recombination to seamlessly stitch blocks of DNA together by mating E. coli in large arrays or pools. This workflow is simpler, cheaper, and higher throughput than current DNA assembly approaches that require DNA to be moved in and out of cells at different procedural steps. We perform over 5,000 assemblies with two to 13 DNA blocks that range from 240 bp to 8 kb and show that SCRIVENER is capable of assembling constructs as long as 23 kb at relatively high throughput and fidelity. Most SCRIVENER errors are deletions between long interspersed repeats. However, SCRIVENER can overcome these errors by enabling assembly and sequence verification at high replication at a nominal additional cost per replicate. We show that SCRIVENER can be used to build combinatorial libraries in arrays or pools, and that DNA blocks onboarded into the platform can be repurposed and reused with any other DNA block in high throughput without a PCR step. Because of these features, DNA engineering with SCRIVENER has the potential to accelerate design-build-test-learn cycles of DNA products.

Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues.

In the absence of perfusable vascular networks, three-dimensional (3D) engineered tissues densely populated with cells quickly develop a necrotic core. Yet the lack of a general approach to rapidly construct such networks remains a major challenge for 3D tissue culture. Here, we printed rigid 3D filament networks of carbohydrate glass, and used them as a cytocompatible sacrificial template in engineered tissues containing living cells to generate cylindrical networks that could be lined with endothelial cells and perfused with blood under high-pressure pulsatile flow. Because this simple vascular casting approach allows independent control of network geometry, endothelialization and extravascular tissue, it is compatible with a wide variety of cell types, synthetic and natural extracellular matrices, and crosslinking strategies. We also demonstrated that the perfused vascular channels sustained the metabolic function of primary rat hepatocytes in engineered tissue constructs that otherwise exhibited suppressed function in their core.

Caulobacter requires a dedicated mechanism to initiate chromosome segregation.

Chromosome segregation in bacteria is rapid and directed, but the mechanisms responsible for this movement are still unclear. We show that Caulobacter crescentus makes use of and requires a dedicated mechanism to initiate chromosome segregation. Caulobacter has a single circular chromosome whose origin of replication is positioned at one cell pole. Upon initiation of replication, an 8-kb region of the chromosome containing both the origin and parS moves rapidly to the opposite pole. This movement requires the highly conserved ParABS locus that is essential in Caulobacter. We use chromosomal inversions and in vivo time-lapse imaging to show that parS is the Caulobacter site of force exertion, independent of its position in the chromosome. When parS is moved farther from the origin, the cell waits for parS to be replicated before segregation can begin. Also, a mutation in the ATPase domain of ParA halts segregation without affecting replication initiation. Chromosome segregation in Caulobacter cannot occur unless a dedicated parS guiding mechanism initiates movement.

Movement control strategies during jumping in a lizard (Anolis valencienni).

Whereas maximal performance is subjected to specific control criteria, sub-maximal movements theoretically allow for an infinite number of control strategies. Yet sub-maximal movements are predominant in the locomotor repertoire of most organisms and often little understood. Previous data on sub-maximal vertical jumping in humans has suggested that a movement effectiveness criterion might best explain the observed control strategy employed. Here we test the generality of this criterion in jumping by inducing lizards to jump both at a range of distances as well as a range of take-off angles. Our results show that while movement effectiveness appears to best explain jumping for different take-off angles, a 'push harder' strategy (i.e. mostly increasing the force output of the system), is used in the control of distance jumping. Thus, our data support the generality of the movement effectiveness criterion for vertical jumping, but not for distance jumping. Sub-maximal distance jumping in the lizard Anolis valencienni appears to be governed by a relatively simple control strategy that allows a rapid response. This accords well to the ecological circumstances in which long jumps are typically used (escape from predators).

The evolution of jumping performance in Caribbean Anolis lizards: solutions to biomechanical trade-offs.

Adaptationist theory predicts that species will evolve functional specializations for occupying different ecological niches. However, whereas performance traits are often complex, most comparative functional studies examine only simple measures of performance (e.g., sprint speed). Here we examine multiple facets of jumping biomechanics in 12 species of Caribbean Anolis lizards. These 12 species represent six ecomorphs, which are distinct ecological and morphological entities that have independently evolved on different Caribbean islands. We first show that the optimal angles for jumping maximum horizontal distances range from 39 degrees to 42 degrees, but the average jump angle of the 12 species is about 36 degrees. Interestingly, these "suboptimal" jumping angles result in only a small decrement in jump distance but substantial savings in flight duration and jump height. Further, our data show that the two key variables associated with increased jumping velocity (hindlimb length and takeoff acceleration) are independent of one another. Thus, there are two possible ways to achieve superior jumping capabilities: to evolve more muscular limbs--as stronger legs will produce more force and, hence, more acceleration--or evolve longer limbs. Our data show that anole species face trade-offs that prevent them from simultaneously optimizing different aspects of jumping ability but that they appear to have evolved behaviors that partially overcome these trade-offs.

In vitro fertilization and artificial activation of eggs of the direct-developing anuran Eleutherodactylus coqui.

Although much is known about the reproductive biology of pond-breeding frogs, there is comparatively little information about terrestrial-breeding anurans, a highly successful and diverse group. This study investigates the activation and in vitro fertilization of eggs of the Puerto Rican coqui frog obtained by hormonally induced ovulation. We report that spontaneous activation occurs in 34% of eggs, probably in response to mechanical stress during oviposition. Artificial activation, as evidenced by the slow block to polyspermy and the onset of zygote division, was elicited both by mechanical stimulation and calcium ionophore exposure in 64% and 83% of the cases, respectively. Finally, one in vitro fertilization protocol showed a 27% success rate, despite the fact that about one third of all unfertilized eggs obtained by hormone injection auto-activate. We expect these findings to aid in the conservation effort of Eleutherodactylus frogs, the largest vertebrate genus.

Induced ovulation and egg deposition in the direct developing anuran Eleutherodactylus coqui.

This study investigates ovulation and egg deposition behaviors in the anuran Eleutherodactylus coqui from Puerto Rico in response to stimulation with gonadotropin and gonadotropin releasing hormones. Five hormones were tested by injection over a range of doses, including mammalian LHRH, avian LHRH, fish LHRH, D-Ala6, des-Gly10 ethylamide LHRH and hCG. We report a low level of ovulation and egg deposition in response to all hormones, with the most complete and consistent results from the non-natural D-Ala6, des-Gly10 ethylamide LHRH derivative. To confirm the viability of eggs produced in this manner we performed in vitro fertilization experiments that resulted in the development of normal frogs. Reproductive behaviors in E. coqui are apparently not controlled by a mammalian form of LHRH as reported in other common laboratory anuran species. D-Ala6, des-Gly10 ethylamide LHRH induces ovulation and deposition of mature and fertilizable eggs in E. coqui.

Microfluidics embedded within extracellular matrix to define vascular architectures and pattern diffusive gradients.

Gradients of diffusive molecules within 3D extracellular matrix (ECM) are essential in guiding many processes such as development, angiogenesis, and cancer. The spatial distribution of factors that guide these processes is complex, dictated by the distribution and architecture of vasculature and presence of surrounding cells, which can serve as sources or sinks of factors. To generate temporally and spatially defined soluble gradients within a 3D cell culture environment, we developed an approach to patterning microfluidically ported microchannels that pass through a 3D ECM. Micromolded networks of sacrificial conduits ensconced within an ECM gel precursor solution are dissolved following ECM gelation to yield functional microfluidic channels. The dimensions and spatial layout of channels are readily dictated using photolithographic methods, and channels are connected to external flow via a gasket that also serves to house the 3D ECM. We demonstrated sustained spatial patterning of diffusive gradients dependent on the architecture of the microfluidic network, as well as the ability to independently populate cells in either the channels or surrounding ECM, enabling the study of 3D morphogenetic processes. To highlight the utility of this approach, we generated model vascular networks by lining the channels with endothelial cells and examined how channel architecture, through its effects on diffusion patterns, can guide the location and morphology of endothelial sprouting from the channels. We show that locations of strongest gradients define positions of angiogenic sprouting, suggesting a mechanism by which angiogenesis is regulated in vivo and a potential means to spatially defining vasculature in tissue engineering applications. This flexible 3D microfluidic approach should have utility in modeling simple tissues and will aid in the screening and identification of soluble factor conditions that drive morphogenetic events such as angiogenesis.

Bacterial chromosome organization and segregation.

Bacterial chromosomes are generally approximately 1000 times longer than the cells in which they reside, and concurrent replication, segregation, and transcription/translation of this crowded mass of DNA poses a challenging organizational problem. Recent advances in cell-imaging technology with subdiffraction resolution have revealed that the bacterial nucleoid is reliably oriented and highly organized within the cell. Such organization is transmitted from one generation to the next by progressive segregation of daughter chromosomes and anchoring of DNA to the cell envelope. Active segregation by a mitotic machinery appears to be common; however, the mode of chromosome segregation varies significantly from species to species.

A spindle-like apparatus guides bacterial chromosome segregation.

Until recently, a dedicated mitotic apparatus that segregates newly replicated chromosomes into daughter cells was believed to be unique to eukaryotic cells. Here we demonstrate that the bacterium Caulobacter crescentus segregates its chromosome using a partitioning (Par) apparatus that has surprising similarities to eukaryotic spindles. We show that the C. crescentus ATPase ParA forms linear polymers in vitro and assembles into a narrow linear structure in vivo. The centromere-binding protein ParB binds to and destabilizes ParA structures in vitro. We propose that this ParB-stimulated ParA depolymerization activity moves the centromere to the opposite cell pole through a burnt bridge Brownian ratchet mechanism. Finally, we identify the pole-specific TipN protein as a new component of the Par system that is required to maintain the directionality of DNA transfer towards the new cell pole. Our results elucidate a bacterial chromosome segregation mechanism that features basic operating principles similar to eukaryotic mitotic machines, including a multivalent protein complex at the centromere that stimulates the dynamic disassembly of polymers to move chromosomes into daughter compartments.

Estudio EPRAS: estudio poblacional del riesgo cardiovascular de una población colombiana / EPRAS Study: population-based study of cardiovascular risk in a Colombian population

Objetivos: Describir el comportamiento de los factores de riesgo relacionados con la hipertensión arterial, el consumo declarado de medicamentos y el perfil metabólico en una población de pacientes valorados en el Servicio de Medicina Interna del Hospital Departamental Felipe Suárez de Salamina, Caldas, durante octubre a diciembre de 2012. Materiales y métodos: Se valoraron 400 pacientes hipertensos del servicio de Medicina Interna del Hospital Departamental Felipe Suárez, durante octubre a diciembre de 2012. Se aplicaron formatos de registro y se procesaron en forma descriptiva. Resultados: Se encontró una prevalencia de sedentarismo del 43,7% y de tabaquismo del 9,3%. En un 84,3% de los hipertensos evaluados se encontraron cifras de presión arterial controladas. El 73,3% tenía pobre control de las cifras de colesterol LDL. Los diuréticos fueron el grupo de medicamentos antihipertensivos de mayor consumo (87,2%). Conclusiones: La prevención y el control de los factores de riesgo cardiovascular deben convertirse en una prioridad, en una política pública saludable del sistema de salud, que impacte a la totalidad de la población, con el fin de disminuir la prevalencia ascendente de los denominados grandes síndromes del adulto, responsables de las mayores tasas relacionadas con morbimortalidad en la población general, a través de la promoción de estilos de vida saludables, realización de actividad física regular y detección temprana de factores de riesgo.

Objectives: To describe the behavior of risk factors associated with hypertension, reported consumption of drugs and metabolic profile in a population of patients evaluated in the internal medicine department of the Hospital Departamental Felipe Suárez of Salamina (in Caldas, Colombia) during the period between October and December 2012. Materials and methods: We evaluated 400 patients with hypertension of the internal medicine department of the Hospital Departamental Felipe Suárez during the months of October, November and December 2012. Registration forms were applied and processed in descriptive form. Results: We found a prevalence of sedentarism of 43.7% and smoking of 9.3%. In 84.3% of hypertensive patients evaluated we found controlled blood pressure. 73.3% had poor control of LDL cholesterol levels. The diuretics were the group of anti hypertensive medication with the highest consumption (87.2%). Conclusions: primary prevention and control of cardiovascular risk factors should be turned into a priority, healthy public policy in the health system, which impacts the entire population, in order to decrease the rising prevalence of so-called large adult syndromes and those which are responsible for the higher rates associated with morbidity and mortality in the general population through promotion of healthy lifestyles, physical activity and early detection of risk factors.

Estudio EDECO Estudio poblacional de deterioro cognitivo en población colombiana / EDECO Study Population study of cognitive impairment in a colombian community

Objetivo: se presenta un estudio poblacional de deterioro cognitivo de un grupo de ancianos valorados en la comunidad en los cuales se determinó la prevalencia de deterioro cognitivo a través de la prueba del reloj. Material y métodos: se valoraron 803 ancianos que habitaban en comunidad, durante los meses de noviembre de 2011 a agosto de 2012, de distintas regiones del país. Se aplicaron formatos de registro y se procesaron en forma descriptiva. Resultados: los relojes evaluados por medio de los criterios de Cacho para detección de deterioro cognitivo, permitieron identificar que 40% de la población de ancianos incluidos en el estudio, presentaban un resultado sugestivo de deterioro cognitivo. El rango de edad más afectado por deterioro cognitivo, en el grupo de relojes calificados, se observó entre los 70 y 79 años (46.1%), seguido por los individuos entre los 80 y los 89 años (31.8%). El rango de edad entre 60 y 69 años mostró una prevalencia de deterioro cognitivo en 19.6%. El 57.6% de los ancianos con resultados sugestivos de deterioro cognitivo presentaban antecedente de hipertensión arterial. El 14.3% de los ancianos evaluados refirieron haber sido diagnosticado con diabetes mellitus, de los cuales 48.7% presentaron hallazgos subjetivos de deterioro cognitivo (17.4% del total de población con deterioro cognitivo). El antecedente de accidente cerebrovascular se observó en el 4.9% de la población, de los cuales 56% obtuvieron puntajes sugestivos de deterioro cognitivo. Conclusiones: con respecto a nuestro estudio, 40% de la población presentaba hallazgos sugestivos de deterioro cognitivo, además se logró evidenciar una importante prevalencia de deterioro cognitivo en factores de riesgo reportados en la literatura. También se observó que en todos los grupos evaluados los errores en la localización de las manecillas predominaron como el principal error en la realización de la prueba, seguido de las alteraciones en la presencia y secuencia de los números, mientras que la realización de la esfera fue la variable menos afectada en todos los grupos poblacionales. Se observó en el estudio que los resultados sugestivos de deterioro cognitivo presentaron una relación directa con el analfabetismo, el nivel educativo y la edad. Finalmente podemos sugerir que la prueba del reloj, en el contexto del anciano colombiano que habita en comunidad, constituye una herramienta útil para el tamizaje de la demencia en atención primaria y geriatría clínica.

Objective: a population-based study of cognitive impairment in a group of elders evaluated in the community in which the prevalence of cognitive impairment was determined through the clock test, is presented. Materials and methods: 803 elderly people living in community during the months of November 2011 to August 2012 from different regions of the country were evaluated. Registration forms were applied and processed in a descriptive way. Results: clocks evaluated by Cacho criteria for detection of cognitive impairment helped to identify that 40% of the elderly population included in the study had a suggestive result of cognitive impairment. The age range most affected by cognitive decline in the group of qualified clocks was observed between 70 and 79 years (46.1%), followed by individuals between 80 and 89 years (31.8%). The age range between 60 and 69 years showed a prevalence of cognitive impairment in 19.6%. 57.6% of the elderly with results suggestive of cognitive impairment had a history of hypertension. 14.3% of the elderly evaluated reported having been diagnosed with diabetes mellitus, and of these, 48.7% had subjective findings of cognitive impairment (17.4% of total population with cognitive impairment). A history of stroke was observed in 4.9% of the population, of which 56% obtained scores suggestive of cognitive impairment. Conclusions: regarding our study, 40% of the population was suggestive of cognitive impairment and additionally a significant prevalence of cognitive impairment in risk factors reported in the literature was evidenced. It was also observed that in all groups evaluated, the errors in locating the hands predominated as the main error in performing the test, followed by alterations in the presence and sequence of numbers while making the circle was the less affected variable in all the population groups. It was observed in the study that the results suggestive of cognitive impairment had a direct relationship with illiteracy, educational level and age. Finally, we can suggest that the clock test in the context of the Colombian elderly living in community is a useful tool for the screening of dementia in primary care and clinical geriatrics.

A biomechanical analysis of intra- and interspecific scaling of jumping and morphology in Caribbean Anolis lizards.

Scaling models predict how functional variables change as animals grow or increase in size evolutionarily. However, few experimental studies have found support for the predictions of these models. Here, we use a force plate to investigate the scaling of functional variables associated with jumping within (for three species) and across adults of 12 species of Anolis lizards. Both ontogenetically (with the exception of Anolis carolinensis) and across the 12 species examined, limb dimensions increased geometrically, making Anolis lizards an ideal study system to test the predictions of geometric scaling models. However, both the ontogenetic and interspecific scaling of functional variables deviated in several aspects from model predictions. Unexpectedly, the scaling of functional variables such as acceleration differed for different species. Whereas acceleration capacity increases with hindlimb length for A. carolinensis, no relationship was detected for the other two species. Interspecifically, the inclusion of two large species in our analysis appears to drive the absence of a correlation between acceleration capacity and hindlimb length across species. These data suggest that selection for enhanced jumping performance is relaxed in larger anoles and support the notion that no scaling model seems to be able to comprehensively predict changes in function with size across species; rather, natural selection seems to drive changes in the scaling relationships of some key variables such as force output or acceleration capacity.