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Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells.

It is impractical to develop a new parts collection for every potential host organism. It is well-established that gene expression parts, like genes, are qualitatively transferable, but there is little quantitative information defining transferability. Here, we systematically quantified the behavior of a parts set across multiple hosts. To do this, we developed a broad host range (BHR) plasmid system compatible with the large, modular CIDAR parts collection forE. coli. This enabled testing of a library of DNA constructs across the Pseudomonadota –Escherichia coli, Pseudomonas putida, Cupriavidus necator, andKomagataeibacter nataicola. Part performance was evaluated with a standardized characterization procedure that quantified expression in terms of molecules of equivalent fluorescein (MEFL), an objective unit of measure. The results showed that the CIDAR parts enable graded gene expression across all organisms – meaning that the same parts can be used to programE. coli, P. putida, C. necator, andK. nataicola. Most parts had a similar expression trend across hosts, although each organism had a different average gene expression level. The variability is enough that to achieve the same MEFL in a different organism, a lookup table is required to translate a design from one host to another. To identify truly divergent parts, we applied linear regression to a combinatorial set of promoters and ribosome binding sites, finding that the promoter J23100 behaves very differently inK. nataicolathan in the other hosts. Thus, it is now possible to evaluate any CIDAR compatible part in three other hosts of interest, and the diversity of these hosts implies that the collection will also be compatible with many other Proteobacteria (Pseudomonadota). Furthermore, this work defines an approach to generalize modular synthetic biology parts sets beyond a single host, making it possible to create a small number of parts sets that can span the tree of life. This will accelerate current efforts to engineer diverse species for environmental, biotechnological, and health applications.

The Synthetic Biology Knowledge System (SBKS) is an instance of the SynBioHub repository that includes text and data information that has been mined from papers published in ACS Synthetic Biology. This paper describes the SBKS curation framework that is being developed to construct the knowledge stored in this repository. The text mining pipeline performs automatic annotation of the articles using natural language processing techniques to identify salient content such as key terms, relationships between terms, and main topics. The data mining pipeline performs automatic annotation of the sequences extracted from the supplemental documents with the genetic parts used in them. Together these two pipelines link genetic parts to papers describing the context in which they are used. Ultimately, SBKS will reduce the time necessary for synthetic biologists to find the information necessary to complete their designs.

Scientific articles contain a wealth of information about experimental methods and results describing biological designs. Due to its unstructured nature and multiple sources of ambiguity and variability, extracting this information from text is a difficult task. In this paper, we describe the development of the synthetic biology knowledge system (SBKS) text processing pipeline. The pipeline uses natural language processing techniques to extract and correlate information from the literature for synthetic biology researchers. Specifically, we apply named entity recognition, relation extraction, concept grounding, and topic modeling to extract information from published literature to link articles to elements within our knowledge system. Our results show the efficacy of each of the components on synthetic biology literature and provide future directions for further advancement of the pipeline.

As an engineering endeavor, synthetic biology requires effective sharing of genetic design information that can be reused in the construction of new designs. While there are a number of large community repositories of design information, curation of this information has been limited. This in turn limits the ways in which design information can be put to use. The aim of this work was to improve this situation by creating a curated library of parts from the International Genetically Engineered Machines (iGEM) registry data set. To this end, an analysis of the Synthetic Biology Open Language (SBOL) version of the iGEM registry was carried out using four different approaches-simple statistics, SnapGene autoannotation, SYNBICT autoannotation, and expert analysis-the results of which are presented herein. Key challenges encountered include the use of free text, insufficient part provenance, part duplication, lack of part removal, and insufficient continuous curation. On the basis of these analyses, the focus has shifted from the creation of a curated iGEM part library to instead the extraction of a set of lessons, which are presented here. These lessons can be exploited to facilitate the creation and curation of other part libraries using a simpler and less labor intensive process.

Biologically derived materials have numerous applications in biomedical fields and beyond. Yet, the properties of these materials are difficult to alter because the natural biosynthetic mechanisms are difficult to elucidate, imitate, or adjust. Thus, many bio-derived materials are isolated from natural tissues, or substitutes are recombinantly produced and then modified ex vivo. A major shift in this paradigm is being driven by the maturing field of synthetic biology, which is innovating a ‘tool box’ of methods to tune biomolecules and biosynthetic mechanisms in vivo. In the context of materials, this has led to higher production titers due to reprogrammed natural biosynthesis and has enabled design of novel materials by combining desired domains in vivo. Here, we highlight recent applications of synthetic biology to bio-derived ribosomal and nonribosomal polymer materials for biomedical applications. We then describe state-of-the-art techniques that will impact bio-derived material production and engineering in the near future. Continued innovation at the convergence of synthetic biology and materials science promises to begin a new era of biomaterial design and synthesis.

Plant-derived secondary metabolites have many applications as fragrances, food additives, dyes, and bioactive medicinal compounds. Production of these compounds in plant cell culture has many advantages, and metabolic engineering strategies have been developed to increase yields. Yet, long-term subculturing can reduce productivity due to epigenetic downregulation of secondary metabolism. Innovative techniques that take advantage of new insights into epigenetic regulation have been developed for a variety of eukaryotes. Applying this epigenetic control to plant cell culture could restore and maintain high productivity, even in cultures that have been in suspension for many years. Here, we highlight current strategies for plant metabolic engineering and review the current understanding of epigenetic regulation in plants, with particular emphasis on applications to secondary metabolic engineering. We then discuss work in the emerging field of epigenetic engineering and postulate how epigenetic engineering techniques developed in mammalian systems could be used to overcome current limitations for industrial production of natural products in plant systems.

Yeast genomes can be assembled from sequencing data, but genome integrations and episomal plasmids often fail to be resolved with accuracy, completeness, and contiguity. Resolution of these features is critical for many synthetic biology applications, including strain quality control and identifying engineering in unknown samples. Here, we report an integrated workflow, named Prymetime, that uses sequencing reads from inexpensive NGS platforms, assembly and error correction software, and a list of synthetic biology parts to achieve accurate whole genome sequences of yeasts with engineering annotated. To build the workflow, we first determined which sequencing methods and software packages returned an accurate, complete, and contiguous genome of an engineered S. cerevisiae strain with two similar plasmids and an integrated pathway. We then developed a sequence feature annotation step that labels synthetic biology parts from a standard list of yeast engineering sequences or from a custom sequence list. We validated the workflow by sequencing a collection of 15 engineered yeasts built from different parent S. cerevisiae and nonconventional yeast strains. We show that each integrated pathway and episomal plasmid can be correctly assembled and annotated, even in strains that have part repeats and multiple similar plasmids. Interestingly, Prymetime was able to identify deletions and unintended integrations that were subsequently confirmed by other methods. Furthermore, the whole genomes are accurate, complete, and contiguous. To illustrate this clearly, we used a publicly available S. cerevisiae CEN.PK113 reference genome and the accompanying reads to show that a Prymetime genome assembly is equivalent to the reference using several standard metrics. Finally, we used Prymetime to resequence the nonconventional yeasts Y. lipolytica Po1f and K. phaffii CBS 7435, producing an improved genome assembly for each strain. Thus, our workflow can achieve accurate, complete, and contiguous whole genome sequences of yeast strains before and after engineering. Therefore, Prymetime enables NGS-based strain quality control through assembly and identification of engineering features.

Pharmaceutical production hosts may be derived from almost any organism, from Chinese Hamster Ovary (CHO) cell lines to isolated actinomycetes. Each host can be improved, historically only through adaptive evolution. Recently, the maturation of organism engineering has expanded the available models, methods, and tools for altering host phenotypes. New tools like CRISPR-associated endonucleases promise to enable precise cellular reprogramming and to access previously intractable hosts. In this review, we discuss the most recent advances in engineering several types of pharmaceutical production hosts. These include model organisms, potential platform hosts with advantageous metabolism or physiology, specialized producers capable of unique biosynthesis, and CHO, the most widely used recombinant protein production host. To realize improved engineered hosts, an increasing number of approaches involving DNA sequencing and synthesis, host rewriting technologies, computational methods, and organism engineering strategies must be used. Integrative workflows that enable application of the right combination of methods to the right production host could enable economical production solutions for emerging human health treatments.

Yeast whole genome sequencing (WGS) lacks end-to-end workflows that identify genetic engineering. Here we present Prymetime, a tool that assembles yeast plasmids and chromosomes and annotates genetic engineering sequences. It is a hybrid workflow—it uses short and long reads as inputs to perform separate linear and circular assembly steps. This structure is necessary to accurately resolve genetic engineering sequences in plasmids and the genome. We show this by assembling diverse engineered yeasts, in some cases revealing unintended deletions and integrations. Furthermore, the resulting whole genomes are high quality, although the underlying assembly software does not consistently resolve highly repetitive genome features. Finally, we assemble plasmids and genome integrations from metagenomic sequencing, even with 1 engineered cell in 1000. This work is a blueprint for building WGS workflows and establishes WGS-based identification of yeast genetic engineering., The cost and complexity of whole genome sequencing limits its use in identifying and validating sequences used for genetic engineering and synthetic biology. Here the authors present Prymetime, an integrated workflow to sequence engineered strains and identify engineering in metagenomes.

International audience; Fingertip haptic feedback offers advantages in many applications , including robotic teleoperation, gaming, and training. However, fingertip size and shape vary significantly across humans, making it difficult to design fingertip interfaces and rendering techniques suitable for everyone. This paper starts with an existing data-driven haptic rendering algorithm that ignores fingertip size, and it then develops two software-based approaches to personalize this algorithm for fingertips of different sizes using either additional data or geometry. We evaluate our algorithms in the rendering of pre-recorded tactile sensations onto rubber casts of six different fingertips as well as onto the real fingertips of 13 human participants. Results on the casts show that both approaches significantly improve performance, reducing force error magnitudes by an average of 78% with respect to the standard non-personalized rendering technique. Congruent results were obtained for real fingertips, with subjects rating each of the two personalized rendering techniques significantly better than the standard non-personalized method.

Existing fingertip haptic devices can deliver different subsets of tactile cues in a compact package, but we have not yet seen a wearable six-degree-of-freedom (6-DOF) display. This paper presents the Fuppeteer (short for Fingertip Puppeteer), a device that is capable of controlling the position and orientation of a flat platform, such that any combination of normal and shear force can be delivered at any location on any human fingertip. We build on our previous work of designing a parallel continuum manipulator for fingertip haptics by presenting a motorized version in which six flexible Nitinol wires are actuated via independent roller mechanisms and proportional-derivative controllers. We evaluate the settling time and end-effector vibrations observed during system responses to step inputs. After creating a six-dimensional lookup table and adjusting simulated inputs using measured Jacobians, we show that the device can make contact with all parts of the fingertip with a mean error of 1.42 mm. Finally, we present results from a human-subject study. A total of 24 users discerned 9 evenly distributed contact locations with an average accuracy of 80.5%. Translational and rotational shear cues were identified reasonably well near the center of the fingertip and more poorly around the edges.

Recently, semirational approaches that rely on combinatorial assembly of characterized DNA components have been used to engineer biosynthetic pathways. In practice, however, it is not practical to assemble and test millions of pathway variants in order to elucidate how different DNA components affect the behavior of a pathway. To address this challenge, we apply a rigorous mathematical approach known as design of experiments (DOE) that can be used to construct empirical models of system behavior without testing all variants. To support this approach, we have developed a tool named Double Dutch, which uses a formal grammar and heuristic algorithms to automate the process of DOE library design. Compared to designing by hand, Double Dutch enables users to more efficiently and scalably design libraries of pathway variants that can be used in a DOE framework and uniquely provides a means to flexibly balance design considerations of statistical analysis, construction cost, and risk of homologous recombination, thereby demonstrating the utility of automating decision making when faced with complex design trade-offs.

International audience; Small size and low weight are critical requirements for wearable and portable haptic interfaces, making it essential to work toward the optimization of their sensing and actuation systems. This paper presents a new approach for task-driven design optimization of fingertip cutaneous haptic devices. Given one (or more) target tactile interactions to render and a cutaneous device to optimize, we evaluate the minimum number and best configuration of the device's actuators to minimize the estimated haptic rendering error. First, we calculate the motion needed for the original cutaneous device to render the considered target interaction. Then, we run a principal component analysis (PCA) to search for possible couplings between the original motor inputs, looking also for the best way to reconfigure them. If some couplings exist, we can redesign our cutaneous device with fewer motors, optimally configured to render the target tactile sensation. The proposed approach is quite general and can be applied to different tactile sensors and cutaneous devices. We validated it using a BioTac tactile sensor and custom plate-based 3-DoF and 6-DoF fingertip cutaneous devices, considering six representative target tactile interactions. The algorithm was able to find couplings between each device's motor inputs, proving it to be a viable approach to optimize the design of wearable and portable cutaneous devices. Finally, we present two examples of optimized designs for our 3-DoF fingertip cutaneous device.

Metabolic engineering requires multiple rounds of strain construction to evaluate alternative pathways and enzyme concentrations. Optimizing multigene pathways stepwise or by randomly selecting enzymes and expression levels is inefficient. Here, we apply methods from design of experiments (DOE) to guide the construction of strain libraries from which the maximum information can be extracted without sampling every possible combination. We use Saccharomyces cerevisiae as a host for a novel six-gene pathway to itaconic acid, selected by comparing alternative shunt pathways that bypass the mitochondrial TCA cycle. The pathway is distinctive for the use of acetylating acetaldehyde dehydrogenase to increase cytosolic acetyl-CoA pools, a bacterial enzyme to synthesize citrate in the cytosol, and an itaconic acid exporter. Precise control over the expression of each gene is enabled by a set of promoter-terminator pairs that span a 174-fold range. Two large combinatorial libraries (160 variants, 2.4 Mb and 32 variants, 0.6 Mb) are designed where the expression levels are selected by statistical methods (I-optimal response surface methodology, full factorial, or Plackett-Burman) with the intent of extracting different types of guiding information after the screen. This is applied to the design of a third library (24 variants, 0.5 Mb) intended to alleviate a bottleneck in cis-aconitate decarboxylase (CAD) expression. The top strain produces 815 mg/l itaconic acid, a 4-fold improvement over the initial strain achieved by iteratively balancing pathway expression. Including a methylated product in the total, the strain produces 1.3 g/l combined itaconic acids. Further, a regression analysis of the libraries reveals the optimal expression level of CAD as well as pairwise interdependencies between genes that result in increased titer and purity of itaconic acid. This work demonstrates adapting algorithmic design strategies to guide automated yeast strain construction and learn information after each iteration.

Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

Traditional metabolic pathway engineering rarely considers the influence of molecular transport. Here, we describe the directed evolution of two heterologous transporters, Candida intermedia GXS1 and Scheffersomyces stipitis XUT3. Growth rate on xylose was improved up to 70% by mutant transporter expression. Most mutants were found to exhibit vastly improved V max values and display an increase in high cell density sugar consumption rates. Mixed glucose and xylose fermentations reveal that mutant transporters can alter the diauxic shift dynamics and the simultaneous sugar utilization capacity of the host strain. Analysis of mutations highlights several important residues influencing transporter function including point mutations at F40 of C. intermedia GXS1 and at E538 of S. stipitis XUT3. This work is the first to demonstrate that molecular transporter proteins can be improved for biotechnological applications through directed evolution in yeast.

Autism spectrum disorder (ASD) impacts 1 in 68 children in the U.S., with tremendous individual and societal costs. Technology-aided intervention, more specifically robotic intervention, has gained momentum in recent years due to the inherent affinity of many children with ASD towards technology. In this paper we present a novel robot-mediated intervention system for imitation skill learning, which is considered a core deficit area for children with ASD. The Robot-mediated Imitation Skill Training Architecture (RISTA) is designed in such a manner that it can operate either completely autonomously or in coordination with a human therapist depending on the intervention need. Experimental results are presented from small user studies validating system functionality, assessing user tolerance, and documenting subject performance. Preliminary results show that this novel robotic system draws more attention from the children with ASD and teaches gestures more effectively as compared to a human therapist. While no broad generalized conclusions can be made about the effectiveness of RISTA based on our small user studies, initial results are encouraging and justify further exploration in the future.

Autism Spectrum Disorder (ASD) impacts 1 in 68 children in the U.S. with tremendous consequent cost in terms of care and treatment. Evidence suggests that early intervention is critical for optimal treatment results. Robots have been shown to have great potential to attract attention of children with ASD and can facilitate early interventions on core deficits. In this paper, we propose a robotic platform that mediates imitation skill training for young children with ASD. Imitation skills are considered to be one of the most important skill deficits in children with ASD, which has a profound impact on social communication. While a few previous works have provided methods for single gesture imitation training, the current paper extends the training to incorporate mixed gestures consisting of multiple single gestures during intervention. A preliminary user study showed that the proposed robotic system was able to stimulate mixed gesture imitation in young children with ASD with promising gesture recognition accuracy.

Yeasts serve as an attractive host organism for the production of biofuels from biomass. These organisms are well suited for industrial fermentation and have many advantages in biomass conversion processes. However, many challenges still remain that limit overall production efficiency, especially for the case of converting lignocellulose. This chapter first highlights advances in metabolic engineering, systems biology, and synthetic biology that support the rewiring of yeasts for fuels applications. Next, we address challenges and advances in moving beyond glucose as the sole carbon source. Finally, we address opportunities for the future by describing advances in moving beyond bioethanol as the fuel and describe methodologies to move beyond current limits of rate, titer, and yield.

Increasingly researchers are attempting to develop robotic technologies for children with autism spectrum disorder (ASD). This pilot study investigated the development and application of a novel robotic system capable of dynamic, adaptive, and autonomous interaction during imitation tasks with embedded real-time performance evaluation and feedback. The system was designed to incorporate both a humanoid robot and a human examiner. We compared child performance within system across these conditions in a sample of preschool children with ASD (n = 8) and a control sample of typically developing children (n = 8). The system was well-tolerated in the sample, children with ASD exhibited greater attention to the robotic system than the human administrator, and for children with ASD imitation performance appeared superior during the robotic interaction.

Utilization of exogenous sugars found in lignocellulosic biomass hydrolysates, such as xylose, must be improved before yeast can serve as an efficient biofuel and biochemical production platform. In particular, the first step in this process, the molecular transport of xylose into the cell, can serve as a significant flux bottleneck and is highly inhibited by other sugars. Here we demonstrate that sugar transport preference and kinetics can be rewired through the programming of a sequence motif of the general form G-G/F-XXX-G found in the first transmembrane span. By evaluating 46 different heterologously expressed transporters, we find that this motif is conserved among functional transporters and highly enriched in transporters that confer growth on xylose. Through saturation mutagenesis and subsequent rational mutagenesis, four transporter mutants unable to confer growth on glucose but able to sustain growth on xylose were engineered. Specifically, Candida intermedia gxs1 Phe(38)Ile(39)Met(40), Scheffersomyces stipitis rgt2 Phe(38) and Met(40), and Saccharomyces cerevisiae hxt7 Ile(39)Met(40)Met(340) all exhibit this phenotype. In these cases, primary hexose transporters were rewired into xylose transporters. These xylose transporters nevertheless remained inhibited by glucose. Furthermore, in the course of identifying this motif, novel wild-type transporters with superior monosaccharide growth profiles were discovered, namely S. stipitis RGT2 and Debaryomyces hansenii 2D01474. These findings build toward the engineering of efficient pentose utilization in yeast and provide a blueprint for reprogramming transporter properties.

Molecular transport is a key process in cellular metabolism. This step is often limiting when using a nonnative carbon source, as exemplified by xylose catabolism in Saccharomyces cerevisiae . As a step toward addressing this limitation, this study seeks to characterize monosaccharide transport preference and efficiency. A group of 26 known and putative monosaccharide transport proteins was expressed in a recombinant Saccharomyces cerevisiae host unable to transport several monosaccharides. A growth-based assay was used to detect transport capacity across six different carbon sources (glucose, xylose, galactose, fructose, mannose, and ribose). A mixed glucose-and-xylose cofermentation was performed to determine substrate preference. These experiments identified 10 transporter proteins that function as transporters of one or more of these sugars. Most of these proteins exhibited broad substrate ranges, and glucose was preferred in all cases. The broadest transporters confer the highest growth rates and strongly prefer glucose. This study reports the first molecular characterization of the annotated XUT genes of Scheffersomyces stipitis and open reading frames from the yeasts Yarrowia lipolytica and Debaryomyces hansenii. Finally, a phylogenetic analysis demonstrates that transporter function clusters into three distinct groups. One particular group comprised of D. hansenii XylHP and S. stipitis XUT1 and XUT3 demonstrated moderate transport efficiency and higher xylose preferences.

The general central dogma frames the emergent properties of life, which make biology both necessary and difficult to engineer. In a process engineering paradigm, each biological process stream and process unit is heavily influenced by regulatory interactions and interactions with the surrounding environment. Synthetic biology is developing the tools and methods that will increase control over these interactions, eventually resulting in an integrative synthetic biology that will allow ground-up cellular optimization. In this review, we attempt to contextualize the areas of synthetic biology into three tiers: (1) the process units and associated streams of the central dogma, (2) the intrinsic regulatory mechanisms, and (3) the extrinsic physical and chemical environment. Efforts at each of these three tiers attempt to control cellular systems and take advantage of emerging tools and approaches. Ultimately, it will be possible to integrate these approaches and realize the vision of integrative synthetic biology when cells are completely rewired for biotechnological goals. This review will highlight progress towards this goal as well as areas requiring further research.

Variation in scion physiology caused by rootstock families, scion clones, and the rootstock–scion interactions were studied in loblolly pine (Pinustaeda L.). Ten full-sib families were used as rootstocks, and six scion clones were grafted on the rootstocks in all combinations. The trees were measured during and after the 2nd year after grafting; the traits measured were needle carbohydrate (starch, sucrose, and hexoses) concentrations and needle N, P, K, Ca, and Mg concentrations. These traits were measured in early fall and early spring. There were highly significant differences among scion clones for all traits. Rootstocks significantly affected hexoses in fall, total sugars in spring, P, K, Ca, and Mg in fall, and Ca and Mg in spring. For most traits the largest variance component was within each rootstock-scion combination; next largest were those for scion clones. Variance components for rootstocks were small or negative for most traits, indicating that the scion has more control than the rootstock on scion physiology of young grafted loblolly pine.

Hexose and pentose cofermentation is regarded as one of the chief obstacles impeding economical conversion of lignocellulosic biomass to biofuels. Over time, successful application of traditional metabolic engineering strategy has produced yeast strains capable of utilizing the pentose sugars (especially xylose and arabinose) as sole carbon sources, yet major difficulties still remain for engineering simultaneous, exogenous sugar metabolism. Beyond catabolic pathways, the focus must shift towards non-traditional aspects of cellular engineering such as host molecular transport capability, catabolite sensing and stress response mechanisms. This review highlights the need for an approach termed 'panmetabolic engineering', a new paradigm for integrating new carbon sources into host metabolic pathways. This approach will concurrently optimize the interdependent processes of transport and metabolism using novel combinatorial techniques and global cellular engineering. As a result, panmetabolic engineering is a whole pathway approach emphasizing better pathways, reduced glucose-induced repression and increased product tolerance. In this paper, recent publications are reviewed in light of this approach and their potential to expand metabolic engineering tools. Collectively, traditional approaches and panmetabolic engineering enable the reprogramming of extant biological complexity and incorporation of exogenous carbon catabolism.

Background: While pulmonary hypertension (PHT) contributes significantly to the morbidity and mortality of adults with sickle cell disease (SCD), it has not been studied in pediatric SCD patients. We sought to evaluate the prevalence and significance of PHT in pediatric SCD population. Methods: From May 1999 to July 2004, 123 echocardiograms were performed in SCD patients. Utilizing the tricuspid valve regurgitant jet, right ventricular systolic pressure (RVSP) was estimated in 56 studies (47 patients, aged 0 to 18 years). PHT was defined as RVSP >30 mmHg. Mean steady-state as well as the most recent white blood count (WBC), hemoglobin (Hb) level, reticulocyte count, ferritin level, and number of red cell units transfused in the year prior to the study were noted. In addition, all chest X-ray readings were reviewed to determine the number and type of life-time pulmonary events (effusions, infiltrates, pneumonia, atelectasis or CHF) in each patient. Acute chest syndrome (ACS) was determined to be any effusion, infiltrate or pneumonia. Statistical analysis included Fisher’s exact test and Spearman’s correlation coefficients. Results: Eleven patients were acutely ill at the time of the echocardiogram and were excluded from the analysis. Of the remaining 36 patients, pulmonary hypertension was present in 24 (66%). There was a significant difference in RVSP between younger (0 to 9 years) and older (10 to 18 years) groups of patient (p A significant negative correlation was shown between RVSP and most recent Hb (r = −0.41; p = 0.012) as well as RVSP and the mean steady state platelet count (r = −0.35; p = 0.035). A positive correlation was found between RVSP and the number of ACS episodes (r = 0.37; p = 0.024), RVSP and mean steady state reticulocyte count (r = 0.51; p = 0.002) as well as the most recent reticulocyte count (r = 0.42; p = 0.012). There was however, no correlation found between RVSP and the number of episodes of CHF, collapse or atelectasis separately or grouped together. No correlation was found between RVSP and the number of units transfused or ferritin level. Conclusion: Pulmonary Hypertension is highly prevalent in the pediatric SCD population and its prevalence increases with age. It correlates with indicators of acute and chronic hemolysis as well as the number of ACS events. Our study did not demonstrate correlations between PHT and iron overload indices. Prospective clinical trials monitoring PHT in SCD pediatric patients are indicated.

Blue spruce (Piceapungens Engelm.) seedlings grown in a nursery for 1 to 5 years and seedlings grown from seed in a greenhouse under continuous light for 2 to 6 months were studied to determine (1) time to bud set on transfer to short days, (2) time to bud-break on subsequent transfer to long days, and (3) the anatomy of the dormant shoot apex. Seasonal development of the shoot apex of a single 50-year-old blue spruce was also monitored.Time to but set on transfer to short days decreased after long periods under continuous light. Time to budbreak on subsequent transfer to long days increased with increasing age in nursery- and greenhouse-grown seedlings. The dormant shoot apex became more highly differentiated as the nursery-grown seedlings aged from 1 to 3 years, then showed no significant difference after 3 years of age.The 50-year-old blue spruce initiated many new needles in the current bud before bud scale formation, which began in mid-May. Needle primordia initiation in the new bud began in late June and slowed down in late August. Apical dome diameter increased and decreased concurrently with the increase and decrease in rate of needle primordia initiation.