Publications by authors named "Pamela A Silver"

Coronavirus-infected cells contain double-membrane vesicles (DMVs) that are key for viral RNA replication and transcription, perforated by hexameric pores connecting the vesicular lumen to the cytoplasm. How pores form and traverse two membranes, and how DMVs organize RNA synthesis, is unknown. Using structure prediction and functional assays, we show that the nonstructural viral membrane protein nsp4 is the key pore organizer, spanning the double membrane and forming most of the pore lining.

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The Cold Spring Harbor Laboratory (CSHL) Summer Course on Synthetic Biology, established in 2013, has emerged as a premier platform for immersive education and research in this dynamic field. Rooted in CSHL's rich legacy of biological discovery, the course offers a comprehensive exploration of synthetic biology's fundamentals and applications. Led by a consortium of faculty from diverse institutions, the course structure seamlessly integrates practical laboratory sessions, exploratory research rotations, and enriching seminars by leaders in the field.

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Tardigrades, microscopic animals that survive a broad range of environmental stresses, express a unique set of proteins termed tardigrade-specific intrinsically disordered proteins (TDPs). TDPs are often expressed at high levels in tardigrades upon desiccation, and appear to mediate stress adaptation. Here, we focus on the proteins belonging to the secreted family of tardigrade proteins termed secretory-abundant heat soluble ("SAHS") proteins, and investigate their ability to protect diverse biological structures.

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Lanthanides, a series of 15 f-block elements, are crucial in modern technology, and their purification by conventional chemical means comes at a significant environmental cost. Synthetic biology offers promising solutions. However, progress in developing synthetic biology approaches is bottlenecked because it is challenging to measure lanthanide binding with current biochemical tools.

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Microbial bioremediation can provide an environmentally friendly and scalable solution to treat contaminated soil and water. However, microbes have yet to optimize pathways for degrading persistent anthropogenic pollutants, in particular organohalides. In this work, we first expand our repertoire of enzymes useful for bioremediation.

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Microorganisms are a promising means to address many societal sustainability challenges owing to their ability to thrive in diverse environments and interface with the microscale chemical world via diverse metabolic capacities. Synthetic biology can engineer microorganisms by rewiring their regulatory networks or introducing new functionalities, enhancing their utility for target applications. In this Review, we provide a broad, high-level overview of various research efforts addressing sustainability challenges through synthetic biology, emphasizing foundational microbiological research questions that can accelerate the development of these efforts.

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Article Synopsis
  • - The increase in greenhouse gases, primarily CO2, is a major threat to the planet, causing climate tipping points that lead to biodiversity loss, global warming, and climate migration.
  • - Global efforts to reduce CO2 emissions must be combined with carbon dioxide removal (CDR) strategies, which could be accelerated using microbes to speed up natural processes and generate valuable by-products.
  • - Research on enhancing silicate weathering using the mineral olivine shows promise for effective CDR, and there is potential for large-scale applications similar to those in existing industries that utilize microbial consortia.
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In gene therapy, potential integration of therapeutic transgene into host cell genomes is a serious risk that can lead to insertional mutagenesis and tumorigenesis. Viral vectors are often used as the gene delivery vehicle, but they are prone to undergoing integration events. More recently, non-viral delivery of linear DNAs having modified geometry such as closed-end linear duplex DNA (CELiD) have shown promise as an alternative, due to prolonged transgene expression and less cytotoxicity.

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Biomolecular condensates formed by liquid-liquid phase separation have been implicated in multiple diseases. Modulation of condensate dynamics by small molecules has therapeutic potential, but so far, few condensate modulators have been disclosed. The SARS-CoV-2 nucleocapsid (N) protein forms phase-separated condensates that are hypothesized to play critical roles in viral replication, transcription, and packaging, suggesting that N condensation modulators might have anti-coronavirus activity across multiple strains and species.

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Microbes can provide a more sustainable and energy-efficient method of food and nutrient production compared to plant and animal sources, but energy-intensive carbon (e.g., sugars) and nitrogen (e.

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Natural systems use weak interactions and avidity effects to give biological systems high specificity and signal-to-noise ratios. Here we describe design principles for engineering fusion proteins that target therapeutic fusion proteins to membrane-bound signaling receptors by first binding to designer-chosen co-receptors on the same cell surface. The key design elements are separate protein modules, one that has no signaling activity and binds to a cell surface receptor with high affinity and a second that binds to a receptor with low or moderate affinity and carries out a desired signaling or inhibitory activity.

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Suppression of the host innate immune response is a critical aspect of viral replication. Upon infection, viruses may introduce one or more proteins that inhibit key immune pathways, such as the type I interferon pathway. However, the ability to predict and evaluate viral protein bioactivity on targeted pathways remains challenging and is typically done on a single-virus or -gene basis.

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Many organisms can survive extreme conditions and successfully recover to normal life. This extremotolerant behavior has been attributed in part to repetitive, amphipathic, and intrinsically disordered proteins that are upregulated in the protected state. Here, we assemble a library of approximately 300 naturally occurring and designed extremotolerance-associated proteins to assess their ability to protect human cells from chemically induced apoptosis.

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Erythropoietin enhances oxygen delivery and reduces hypoxia-induced cell death, but its pro-thrombotic activity is problematic for use of erythropoietin in treating hypoxia. We constructed a fusion protein that stimulates red blood cell production and neuroprotection without triggering platelet production, a marker for thrombosis. The protein consists of an anti-glycophorin A nanobody and an erythropoietin mutant (L108A).

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At the single-cell level, protein kinase activity is typically inferred from downstream transcriptional reporters. However, promoters are often coregulated by several pathways, making the activity of a specific kinase difficult to deconvolve. Here, we present modular, direct, and specific sensors of bacterial kinase activity, including FRET-based sensors, as well as a synthetic transcription factor based on the lactose repressor (LacI) that has been engineered to respond to phosphorylation.

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Synthetic biology seeks to redesign biological systems to perform novel functions in a predictable manner. Recent advances in bacterial and mammalian cell engineering include the development of cells that function in biological samples or within the body as minimally invasive diagnostics or theranostics for the real-time regulation of complex diseased states. Ex vivo and in vivo cell-based biosensors and therapeutics have been developed to target a wide range of diseases including cancer, microbiome dysbiosis and autoimmune and metabolic diseases.

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Encapsulins are recently discovered protein compartments able to specifically encapsulate cargo proteins in vivo. Encapsulation is dependent on C-terminal targeting peptides (TPs). Here, we characterize and engineer TP-shell interactions in the Thermotoga maritima and Myxococcus xanthus encapsulin systems.

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Abundant links between the gut microbiota and human health indicate that modification of bacterial function could be a powerful therapeutic strategy. The inaccessibility of the gut and inter-connections between gut bacteria and the host make it difficult to precisely target bacterial functions without disrupting the microbiota and/or host physiology. Herein we describe a multidisciplinary approach to modulate the expression of a specific bacterial gene within the gut by oral administration.

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Coupling recent advancements in genetic engineering of diverse microbes and gas-driven fermentation provides a path towards sustainable commodity chemical production. Cupriavidus necator H16 is a suitable species for this task because it effectively utilizes H and CO and is genetically tractable. Here, we demonstrate the versatility of C.

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In synthetic circuits, CRISPR-Cas systems have been used effectively for endpoint changes from an initial state to a final state, such as in logic gates. Here, we use deactivated Cas9 (dCas9) and deactivated Cas12a (dCas12a) to construct dynamic RNA ring oscillators that cycle continuously between states over time in bacterial cells. While our dCas9 circuits using 103-nt guide RNAs showed irregular fluctuations with a wide distribution of peak-to-peak period lengths averaging approximately nine generations, a dCas12a oscillator design with 40-nt CRISPR RNAs performed much better, having a strongly repressed off-state, distinct autocorrelation function peaks, and an average peak-to-peak period length of ∼7.

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Determining where an object has been is a fundamental challenge for human health, commerce, and food safety. Location-specific microbes in principle offer a cheap and sensitive way to determine object provenance. We created a synthetic, scalable microbial spore system that identifies object provenance in under 1 hour at meter-scale resolution and near single-spore sensitivity and can be safely introduced into and recovered from the environment.

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Synthetic biology has promised and delivered on an impressive array of applications based on genetically modified microorganisms. While novel biotechnology undoubtedly offers benefits, like all new technology, precautions should be considered during implementation to reduce the risk of both known and unknown adverse effects. To achieve containment of transgenic microorganisms, confidence to a near-scientific certainty that they cannot transfer their transgenic genes to other organisms, and that they cannot survive to propagate in unintended environments, is a priority.

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