Synthetic genetic circuits to uncover the OCT4 trajectories of successful reprogramming of human fibroblasts.

Reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs) is inefficient, with heterogeneity among transcription factor (TF) trajectories driving divergent cell states. Nevertheless, the impact of TF dynamics on reprogramming efficiency remains uncharted. We develop a system that accurately reports OCT4 protein levels in live cells and use it to reveal the trajectories of OCT4 in successful reprogramming. Our system comprises a synthetic genetic circuit that leverages noise to generate a wide range of OCT4 trajectories and a microRNA targeting endogenous OCT4 to set total cellular OCT4 protein levels. By fusing OCT4 to a fluorescent protein, we are able to track OCT4 trajectories with clonal resolution via live-cell imaging. We discover that a supraphysiological, stable OCT4 level is required, but not sufficient, for efficient iPSC colony formation. Our synthetic genetic circuit design and high-throughput live-imaging pipeline are generalizable for investigating TF dynamics for other cell fate programming applications.

Synthetic genetic circuits to uncover and enforce the OCT4 trajectories of successful reprogramming of human fibroblasts.

Reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs) is inefficient, with heterogeneity among transcription factor (TF) trajectories driving divergent cell states. Nevertheless, the impact of TF dynamics on reprogramming efficiency remains uncharted. Here, we identify the successful reprogramming trajectories of the core pluripotency TF, OCT4, and design a genetic controller that enforces such trajectories with high precision. By combining a genetic circuit that generates a wide range of OCT4 trajectories with live-cell imaging, we track OCT4 trajectories with clonal resolution and find that a distinct constant OCT4 trajectory is required for colony formation. We then develop a synthetic genetic circuit that yields a tight OCT4 distribution around the identified trajectory and outperforms in terms of reprogramming efficiency other circuits that less accurately regulate OCT4. Our synthetic biology approach is generalizable for identifying and enforcing TF dynamics for cell fate programming applications.

The sound of silence: Transgene silencing in mammalian cell engineering.

To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene circuits within native transcriptional networks. The utility of synthetic gene circuits for cell engineering relies on the ability to control the expression of all constituent transgene components. Transgene silencing, defined as the loss of expression over time, persists as an obstacle for engineering primary cells and stem cells with transgenic cargos. In this review, we highlight the challenge that transgene silencing poses to the robust engineering of mammalian cells, outline potential molecular mechanisms of silencing, and present approaches for preventing transgene silencing. We conclude with a perspective identifying future research directions for improving the performance of synthetic gene circuits.

PERSIST platform provides programmable RNA regulation using CRISPR endoRNases.

Regulated transgene expression is an integral component of gene therapies, cell therapies and biomanufacturing. However, transcription factor-based regulation, upon which most applications are based, suffers from complications such as epigenetic silencing that limit expression longevity and reliability. Constitutive transgene transcription paired with post-transcriptional gene regulation could combat silencing, but few such RNA- or protein-level platforms exist. Here we develop an RNA-regulation platform we call "PERSIST" which consists of nine CRISPR-specific endoRNases as RNA-level activators and repressors as well as modular OFF- and ON-switch regulatory motifs. We show that PERSIST-regulated transgenes exhibit strong OFF and ON responses, resist silencing for at least two months, and can be readily layered to construct cascades, logic functions, switches and other sophisticated circuit topologies. The orthogonal, modular and composable nature of this platform as well as the ease in constructing robust and predictable gene circuits promises myriad applications in gene and cell therapies.

Principles for the design of multicellular engineered living systems.

Remarkable progress in bioengineering over the past two decades has enabled the formulation of fundamental design principles for a variety of medical and non-medical applications. These advancements have laid the foundation for building multicellular engineered living systems (M-CELS) from biological parts, forming functional modules integrated into living machines. These cognizant design principles for living systems encompass novel genetic circuit manipulation, self-assembly, cell-cell/matrix communication, and artificial tissues/organs enabled through systems biology, bioinformatics, computational biology, genetic engineering, and microfluidics. Here, we introduce design principles and a blueprint for forward production of robust and standardized M-CELS, which may undergo variable reiterations through the classic design-build-test-debug cycle. This Review provides practical and theoretical frameworks to forward-design, control, and optimize novel M-CELS. Potential applications include biopharmaceuticals, bioreactor factories, biofuels, environmental bioremediation, cellular computing, biohybrid digital technology, and experimental investigations into mechanisms of multicellular organisms normally hidden inside the "black box" of living cells.

Cost-effectiveness analysis of multimodal prognostication in cardiac arrest with EEG monitoring.

OBJECTIVE: To determine cost-effectiveness parameters for EEG monitoring in cardiac arrest prognostication. METHODS: We conducted a cost-effectiveness analysis to estimate the cost per quality-adjusted life-year (QALY) gained by adding continuous EEG monitoring to standard cardiac arrest prognostication using the American Academy of Neurology Practice Parameter (AANPP) decision algorithm: neurologic examination, somatosensory evoked potentials, and neuron-specific enolase. We explored lifetime cost-effectiveness in a closed system that incorporates revenue back into the medical system (return) from payers who survive a cardiac arrest with good outcome and contribute to the health system during the remaining years of life. Good outcome was defined as a Cerebral Performance Category (CPC) score of 1-2 and poor outcome as CPC of 3-5. RESULTS: An improvement in specificity for poor outcome prediction of 4.2% would be sufficient to make continuous EEG monitoring cost-effective (baseline AANPP specificity = 83.9%). In sensitivity analysis, the effect of increased sensitivity on the cost-effectiveness of EEG depends on the utility (u) assigned to a poor outcome. For patients who regard surviving with a poor outcome (CPC 3-4) worse than death (u = -0.34), an increased sensitivity for poor outcome prediction of 13.8% would make AANPP + EEG monitoring cost-effective (baseline AANPP sensitivity = 76.3%). In the closed system, an improvement in sensitivity of 1.8% together with an improvement in specificity of 3% was sufficient to make AANPP + EEG monitoring cost-effective, assuming lifetime return of 50% (USD $70,687). CONCLUSION: Incorporating continuous EEG monitoring into cardiac arrest prognostication is cost-effective if relatively small improvements in sensitivity and specificity are achieved.

Correlates of individual variation in the porphyrin-based fluorescence of red-necked nightjars (Caprimulgus ruficollis).

Many nocturnal animals, including invertebrates such as scorpions and a variety of vertebrate species, including toadlets, flying squirrels, owls, and nightjars, emit bright fluorescence under ultraviolet light. However, the ecological significance of this unique coloration so attached to nocturnality remains obscure. Here, we used an intensively studied population of migratory red-necked nightjars (Caprimulgus ruficollis) to investigate inter-individual variation in porphyrin-based pink fluorescence according to sex, age, body condition, time of the year, and the extent of white plumage patches known to be involved in sexual communication. Males and females exhibited a similar extent of pink fluorescence on the under-side of the wings in both juvenile and adult birds, but males had larger white patches than females. Body condition predicted the extent of pink fluorescence in juvenile birds, but not in adults. On average, the extent of pink fluorescence in juveniles increased by ca. 20% for every 10-g increase in body mass. For both age classes, there was a slight seasonal increase (1-4% per week) in the amount of fluorescence. Our results suggest that the porphyrin-based coloration of nightjars might signal individual quality, at least in their first potential breeding season, although the ability of these and other nocturnal birds to perceive fluorescence remains to be unequivocally proven.

A high-throughput screening and computation platform for identifying synthetic promoters with enhanced cell-state specificity (SPECS).

Cell state-specific promoters constitute essential tools for basic research and biotechnology because they activate gene expression only under certain biological conditions. Synthetic Promoters with Enhanced Cell-State Specificity (SPECS) can be superior to native ones, but the design of such promoters is challenging and frequently requires gene regulation or transcriptome knowledge that is not readily available. Here, to overcome this challenge, we use a next-generation sequencing approach combined with machine learning to screen a synthetic promoter library with 6107 designs for high-performance SPECS for potentially any cell state. We demonstrate the identification of multiple SPECS that exhibit distinct spatiotemporal activity during the programmed differentiation of induced pluripotent stem cells (iPSCs), as well as SPECS for breast cancer and glioblastoma stem-like cells. We anticipate that this approach could be used to create SPECS for gene therapies that are activated in specific cell states, as well as to study natural transcriptional regulatory networks.

The road to opportunities: landscape change promotes body-size divergence in a highly mobile species.

Landscape change provides a suitable framework for investigating population-level responses to novel ecological pressures. However, relatively little attention has been paid to examine the potential influence of landscape change on the geographic scale of population differentiation. Here, we tested for morphological differentiation of red-necked nightjars Caprimulgus ruficollis breeding in a managed property and a natural reserve situated less than 10 km apart. At both sites, we also estimated site fidelity over 5 years and quantified the potential foraging opportunities for nightjars. Breeding birds in the managed habitat were significantly larger in size-as indexed by keel length-than those in the natural one. However, there were no significant differences in wing or tail length. Immigration from neighboring areas was almost negligible and, furthermore, no individual (out of 1130 captures overall) exchanged habitats between years, indicating strong site fidelity. Food supply for nightjars was equally abundant in both habitats, but the availability of foraging sites was remarkably higher in the managed property. As a result, nightjars-particularly fledglings-in the latter habitat benefited from increased foraging opportunities in relation to those in the natural site. It seems likely that the fine-scale variation in nightjar morphology reflects a phenotypic response to unequal local conditions, since non-random dispersal or differential mortality had been determined not to be influential. High site fidelity appears to contribute to the maintenance of body-size differences between the two habitats. Results from this nightjar population highlight the potential of human-induced landscape change to promote population-level responses at exceedingly small geographic scales.

Human-induced changes in landscape configuration influence individual movement routines: lessons from a versatile, highly mobile species.

Landscape conversion by humans may have detrimental effects on animal populations inhabiting managed ecosystems, but human-altered areas may also provide suitable environments for tolerant species. We investigated the spatial ecology of a highly mobile nocturnal avian species-the red-necked nightjar (Caprimulgus ruficollis)-in two contrastingly managed areas in Southwestern Spain to provide management recommendations for species having multiple habitat requirements. Based on habitat use by radiotagged nightjars, we created maps of functional heterogeneity in both areas so that the movements of breeding individuals could be modeled using least-cost path analyses. In both the natural and the managed area, nightjars used remnants of native shrublands as nesting sites, while pinewood patches (either newly planted or natural mature) and roads were selected as roosting and foraging habitats, respectively. Although the fraction of functional habitat was held relatively constant (60.9% vs. 74.1% in the natural and the managed area, respectively), landscape configuration changed noticeably. As a result, least-cost routes (summed linear distances) from nest locations to the nearest roost and foraging sites were three times larger in the natural than in the managed area (mean ± SE: 1356±76 m vs. 439±32 m). It seems likely that the increased proximity of functional habitats in the managed area relative to the natural one is underlying the significantly higher abundances of nightjars observed therein, where breeders should travel shorter distances to link together essential resources, thus likely reducing their energy expenditure and mortality risks. Our results suggest that landscape configuration, but not habitat availability, is responsible for the observed differences between the natural and the managed area in the abundance and movements of breeding nightjars, although no effect on body condition was detected. Agricultural landscapes could be moderately managed to preserve small native remnants and to favor the juxtaposition of functional habitats to benefit those farm species relying on patchy resources.

Atypical primary carcinoid tumour of the skin.

We present a new case of a primary carcinoid tumour of the skin. The mitotic index (4/10 HPF) warrants classification of this case as atypical. The patient was a 58-year-old woman with a 1-year history of a mass on the scalp. Literature review showed this to be only the seventh case of primary carcinoid tumour of the skin. Importantly, the evolution has been favourable in all seven tumours, with a mean follow-up of 2.5 years for the previous six cases. Although the number of cases is too small to draw definitive conclusions, information to date suggests that this type of tumour can be expected to have a benign behaviour, despite the presence in some cases of criteria suggestive of uncertainty, such as the presence of mitosis.

Angiolipoma espinal: descripción de dos casos y revisión de la bibliografía / Angiolipoma involving the spine: a report of two cases and a review of the literature

El angiolipoma espinal es una lesión compuesta por adipocitos maduros con vasos sanguíneos anormales. Hay descritos unos 60 casos, la mayoría en el espacio extradural con predilección por el área dorsal. En nuestro hospital hemos encontrado dos casos documentados con radiología convencional, TC y RM, objeto de esta publicación. Tras revisar la bibliografía y los hallazgos en los diferentes métodos de imagen señalamos la importante aportación de la RM, en especial las técnicas con supresión de la grasa, en el diagnóstico preoperatorio de estas lesiones. En general, presentan alta intensidad de señal en secuencias potenciadas en T1 con algunas áreas de iso/hiposeñal en su interior, que lo diferencia de la lipomatosis que presenta alta intensidad de señal en secuencias ponderadas en T1 pero homogénea. Existe realce tras la administración de contraste paramagnético, menos evidente en las lesiones con muy alta intensidad de señal en secuencias potenciales en T1. En secuencias ponderadas en T2 pueden presentar intensidad de señal variable o alta. Las secuencias con supresión de la grasa aportan especificidad al realzar el componente no lipomatoso (angiomatoso) de la lesión. La asociación de estos hallazgos en RM junto a alteraciones en el esqueleto adyacente en un contexto clínico de compresión medular progresiva debe hacernos pensar en esta lesión, infrecuente y con favorable respuesta al tratamiento quirúrgico. (AU)