Synthetic Genomics (United States)

The performance of dry powder inhalers (DPIs) is governed by a complex interplay of deagglomeration, attachment, detachment, and dispersion. Here, we show that crystal morphology serves as a master variable that elucidates and redefines the mechanistic roles of process parameters in DPI systems. Through the deliberate engineering of four distinct crystal morphologies of fluticasone propionate (FP), we systematically decoupled the effects of blending time (15–120 min) and flow rate (40–80 L/min) on aerosolization. Analysis of the fine particle fraction (FPF) and particle deposition patterns allowed us to isolate the contributions of each parameter to the underlying aerosolization mechanisms. Our results reveal a sophisticated, morphology-dependent interplay. We found that for specific crystal habits, the role of blending time is not confined to modulating deagglomeration and attachment but extends to critically impacting detachment. Similarly, the influence of flow rate is not limited to detachment; for certain morphologies, it becomes a significant factor in driving deagglomeration. Ultimately, this work demonstrates that crystal morphology does not merely affect dispersion; it dictates the extent to which blending and flow rate control the entire cascade of aerosolization events. These findings establish a robust design principle for the rational engineering of inhalable crystals to achieve predictable and optimized therapeutic performance.

ABSTRACTDNA sequencing of complex microbial communities allows for the classification and quantification of thousands of distinct organisms in diverse environmental niches. We present a three domain “Universal Amplicon” (UA) method to simultaneously amplify DNA from the ribosomal small subunit locus from bacteria, archaea, and eukaryotes (and their organelles) using a single pair of amplification primers. We demonstrate the amenability of the UA to multiplexed Illumina library preparation and MiSeq-based sequencing. We validate the UA by sequencing a commercially available microbial community of known quantitative composition and through direct comparison to a shotgun metagenomics dataset. Following validation, we apply the UA to a time-course study of the wetlands of the Northern Imperial Valley in California and show substantial and variable microbial life in the Salton Sea and nearby waters. We demonstrate that the microbial ecology of the Salton Sea varies on at least a monthly basis and is distinct from the surrounding area. Finally, we contribute an open-source Shiny app for real-time analysis of complex metagenomic communities, with application to this study and far beyond.

The development of green energy is important to mitigate global warming. Jatropha (Jatropha curcas L.) is a promising candidate for the production of alternative biofuel, which could reduce the burden on the Earth's resources. Jatropha seeds contain a large quantity of lipids that can be used to produce biofuel, and the rest of the plant has many other uses. Currently, techniques for plant genetic transformation are extensively employed to study, create, and improve the specific characteristics of the target plant. Successful transformation involves the alteration of plants and their genetic materials. The aim of this study was to generate Jatropha plants that can support biofuel production by increasing their seed size using genes found via the rice FOX-hunting system. The present study improved previous protocols, enabling the production of transgenic Jatropha in two steps: the first step involved using auxins and dark incubation to promote root formation in excised shoots and the second step involved delaying the timing of antibiotic selection in the cultivation medium. Transgenic plants were subjected to PCR analysis; the transferred gene expression was confirmed via RT-PCR and the ploidy level was investigated. The results suggest that the genes associated with larger seed size in Arabidopsis thaliana, which were found using the rice FOX-hunting system, produce larger seeds in Jatropha.

Reduced β-cell function and insulin deficiency are hallmarks of diabetes mellitus, which is often accompanied by the malfunction of glucagon-secreting α-cells. While insulin therapy has been developed to treat insulin deficiency, the on-demand supplementation of glucagon for acute hypoglycemia treatment remains inadequate. Here, we describe a transdermal patch that mimics the inherent counterregulatory effects of β-cells and α-cells for blood glucose management by dynamically releasing insulin or glucagon. The two modules share a copolymerized matrix but comprise different ratios of the key monomers to be "dually responsive" to both hyper- and hypoglycemic conditions. In a type 1 diabetic mouse model, the hybrid patch effectively controls hyperglycemia while minimizing the occurrence of hypoglycemia in the setting of insulin therapy with simulated delayed meal or insulin overdose.

The conversion of sunlight into chemical energy by photosynthesis drives life on earth as it provides food and feed, as well as industrial feed stocks for specialized chemicals and biofuels. Photosynthesis is a function of the photosynthetic membrane that forms the thylakoids inside plant chloroplasts. Thylakoid membranes harbor the protein complexes necessary for photosynthetic electron transport and ATP synthesis. These complexes are embedded into a specialized glycerolipid matrix. Two decades of research on the biochemistry of the glycerolipids of the photosynthetic membrane, their transport and remodeling in response to environmental cues and the underlying regulatory processes has provided novel fundamental insights into how photosynthetic membranes are assembled and protected in plant chloroplasts. An overview of the key findings is provided.

A new wave of interest in cell-free protein synthesis (CFPS) systems has shown their utility for producing proteins at high titers, establishing genetic regulatory element libraries ( e.g., promoters, ribosome binding sites) in nonmodel organisms, optimizing biosynthetic pathways before implementation in cells, and sensing biomarkers for diagnostic applications. Unfortunately, most previous efforts have focused on a select few model systems, such as Escherichia coli. Broadening the spectrum of organisms used for CFPS promises to better mimic host cell processes in prototyping applications and open up new areas of research. Here, we describe the development and characterization of a facile CFPS platform based on lysates derived from the fast-growing bacterium Vibrio natriegens, which is an emerging host organism for biotechnology. We demonstrate robust preparation of highly active extracts using sonication, without specialized and costly equipment. After optimizing the extract preparation procedure and cell-free reaction conditions, we show synthesis of 1.6 ± 0.05 g/L of superfolder green fluorescent protein in batch mode CFPS, making it competitive with existing E.coli CFPS platforms. To showcase the flexibility of the system, we demonstrate that it can be lyophilized and retain biosynthesis capability, that it is capable of producing antimicrobial peptides, and that our extract preparation procedure can be coupled with the recently described Vmax Express strain in a one-pot system. Finally, to further increase system productivity, we explore a knockout library in which putative negative effectors of CFPS are genetically removed from the source strain. Our V.natriegens-derived CFPS platform is versatile and simple to prepare and use. We expect it will facilitate expansion of CFPS systems into new laboratories and fields for compelling applications in synthetic biology.

This article is not available for publication.

Polyhydroxyalkanoate (PHA) is a thermoplastic polymer with several advantageous properties, including biomass origin, biocompatibility, and biodegradability. PHA is synthesized in transgenic plants harboring 3 enzymatic genes: phaA, phaB, and phaC (collectively referred to as phaABC). PHA-producing plants exhibit severe growth inhibition that leads to extremely low PHA accumulation when these enzymes are localized in the cytosol. This growth inhibition could be attributed to the deleterious effects of the PHA biosynthetic pathway on endogenous essential metabolites or to PHA cytotoxicity itself. We performed precise morphological observations of phaABC-overexpressing Arabidopsis (ABC-ox), which displayed typical growth inhibition. On growth medium without sucrose, ABC-ox exhibited a pale green phenotype, dwarfism, including small cotyledons and true leaves, and short roots. ABC-ox partially recovered from this growth inhibition when the growth medium was supplemented with 1% sucrose. This recovery was reversed after ABC-ox grown on 1% sucrose medium was transferred to soil. ABC-ox grown on 1% sucrose medium not only demonstrated recovery from growth inhibition but were also the only examined plants with PHA accumulation, suggesting that growth inhibition was not caused by PHA cytotoxicity but rather by a lack of essential metabolites.