Publications by authors named "Robert E. Campbell"

Recombinant optogenetic and chemogenetic proteins are potent tools for manipulating neuronal activity and controlling neural circuit function. However, there are few analogous tools for manipulating the structure of neural circuits. Here, we introduce three rationally designed genetically encoded tools that use E3 ligase-dependent mechanisms to trigger the degradation of synaptic scaffolding proteins, leading to functional ablation of synapses.

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Intense brain activity elevates extracellular potassium, potentially leading to overexcitation and seizures. Astrocytes are crucial for restoring healthy potassium levels, and an emerging focus on their Kir4.1 channels has reopened the quest into the underlying mechanisms.

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Measuring whole-brain distributed functional activity is an important unmet need in neuroscience, requiring high temporal resolution and cellular specificity across large volumes. Functional optoacoustic neuro-tomography (FONT) with genetically encoded calcium ion indicators is a promising approach towards this goal. However, it has not yet been applied in the near-infrared (NIR) range that provides deep penetration and low vascular background optimal for neuroimaging.

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Genetically encoded calcium (Ca) indicators (GECIs) are widely used for imaging neuronal activity, yet current limitations of existing red fluorescent GECIs have constrained their applicability. The inherently dim fluorescence and low signal-to-noise ratio of red-shifted GECIs have posed significant challenges. More critically, several red-fluorescent GECIs exhibit photoswitching when exposed to blue light, thereby limiting their applicability in all-optical experimental approaches.

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The editorial discusses the practice of peer review for SPIE Neurophotonics.

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A chemigenetic indicator with an affinity suitable for imaging of intracellular sodium ions (Na) in mammalian cells was developed. The indicator, based on a chimera of green fluorescent protein (GFP) and HaloTag labeled with a synthetic crown ether chelator, was produced by a combination of rational design and directed evolution. In mammalian cells the indicator exhibited an approximately 100% increase in excitation ratio when the cells were treated with 20 mM Na and an ionophore.

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Potassium ion (K) is the most abundant metal ion in cells and plays an indispensable role in practically all biological systems. Although there have been reports of both synthetic and genetically encoded fluorescent K indicators, there remains a need for an indicator that is genetically targetable, has high specificity for K versus Na, and has a high fluorescent response in the red to far-red wavelength range. Here, we introduce a series of chemigenetic K indicators, designated as the HaloKbp1 series, based on the bacterial K-binding protein (Kbp) inserted into HaloTag7 self-labeled with environmentally sensitive rhodamine derivatives.

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Cells utilize ubiquitin as a posttranslational protein modifier to convey various signals such as proteasomal degradation. The dysfunction of ubiquitylation or following proteasomal degradation can give rise to the accumulation and aggregation of improperly ubiquitylated proteins, which is known to be a general causation of many neurodegenerative diseases. Thus, the characterization of substrate peptide sequences of E3 ligases is crucial in biological and pharmaceutical sciences.

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Synthetic-based fluorescent chemosensors and protein-based fluorescent biosensors are two well-established classes of tools for visualizing and monitoring biological processes in living tissues. Chemigenetic sensors, created using a combination of both synthetic parts and protein parts, are an emerging class of tools that aims to combine the strengths, and overcome the drawbacks, of traditional chemosensors and biosensors. This review will survey the landscape of strategies used for fluorescent chemigenetic sensor design.

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Astrocytes control brain activity via both metabolic processes and gliotransmission, but the physiological links between these functions are scantly known. Here we show that endogenous activation of astrocyte type-1 cannabinoid (CB1) receptors determines a shift of glycolysis towards the lactate-dependent production of D-serine, thereby gating synaptic and cognitive functions in male mice. Mutant mice lacking the CB1 receptor gene in astrocytes (GFAP-CB1-KO) are impaired in novel object recognition (NOR) memory.

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The editorial introduces the Special Section on Molecular Neurophotonics.

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An animal infection model was evaluated on sheep and goats to confirm which species infected with Salmonella enterica serovar Enteritidis C StR (SE13) would provide a consistent and high frequency of Salmonella colonization in lymph nodes (LNs) without causing undue animal morbidity. Sheep and goats (n = 5) were intradermally inoculated with Salmonella, postincubated for 7 days, and euthanized. Superficial cervical, medial iliac, subiliac, mammary, and popliteal LNs were excised from each carcass.

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The development of new or improved single fluorescent protein (FP)-based biosensors (SFPBs), particularly those with excitation and emission at near-infrared wavelengths, is important for the continued advancement of biological imaging applications. In an effort to accelerate the development of new SFPBs, we report modified transposons for the transposase-based creation of libraries of FPs randomly inserted into analyte binding domains, or vice versa. These modified transposons feature ends that are optimized to minimize the length of the linkers that connect the FP to the analyte binding domain.

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Significance: Genetically encoded calcium ion () indicators (GECIs) are powerful tools for monitoring intracellular concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited.

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We developed a system for optogenetic release of single molecules in cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable protein and released them by short pulses of light. Our method allows for a light dose-dependent delivery of functional proteins to the cytosol and plasma membrane in amounts compatible with single-molecule imaging, greatly simplifying access to single-molecule microscopy of any protein in live cells.

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l-Lactate is a monocarboxylate produced during the process of cellular glycolysis and has long generally been considered a waste product. However, studies in recent decades have provided new perspectives on the physiological roles of l-lactate as a major energy substrate and a signaling molecule. To enable further investigations of the physiological roles of l-lactate, we have developed a series of high-performance (Δ/ = 15 to 30 ), intensiometric, genetically encoded green fluorescent protein (GFP)-based intracellular l-lactate biosensors with a range of affinities.

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Fluorescent imaging sensors based on genetically-encoded and biocompatible proteins have become important tools in medical and biological research due to their high spatiotemporal resolution and ease of use. Protein engineering has led to the development of imaging sensors that visualize changes in the concentration of various target molecules/ions, such as calcium ions. In addition, the development of chemigenetic sensors based on complexes of proteins and synthetic molecules has been gaining momentum in recent years.

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Genetically encoded sensors enable quantitative imaging of analytes in live cells. Sensors are commonly constructed by combining ligand-binding domains with one or more sensitized fluorescent protein (FP) domains. Sensors based on a single FP can be susceptible to artifacts caused by changes in sensor levels or distribution in vivo.

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Significance: Genetically encoded calcium ion (Ca) indicators (GECIs) are powerful tools for monitoring intracellular Ca concentration changes in living cells and model organisms. In particular, GECIs have found particular utility for monitoring the transient increase of Ca concentration that is associated with the neuronal action potential. However, the palette of highly optimized GECIs for imaging of neuronal activity remains relatively limited.

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L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intra-cellular dynamics of L-lactate are currently hampered by the limited selection and performance of L-lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular L-lactate biosensor, designated eLACCO2.

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The development of a new materials platform capable of sustaining the functionality of proteinous sensor molecules over an extended period without being affected by biological contaminants in living systems, such as proteases, is highly demanded. In this study, our primary focus was on fabricating new core-shell fibremats using unique polymer materials, capable of functionalizing encapsulated sensor proteins while resisting the effects of proteases. The core-fibre parts of core-shell fibremats were made using a newly developed post-crosslinkable water-soluble copolymer, poly(2-hydroxypropyl methacrylamide)--poly(diacetone methacrylamide), and the bifunctional crosslinking agent, adipic dihydrazide, while the shell layer of the nanofibers was made of nylon 6.

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Genetically encoded pH sensors based on fluorescent proteins are valuable tools for the imaging of cellular events that are associated with pH changes, such as exocytosis and endocytosis. Superecliptic pHluorin (SEP) is a pH-sensitive green fluorescent protein (GFP) variant widely used for such applications. Here, we report the rational design, development, structure, and applications of Lime, an improved SEP variant with higher fluorescence brightness and greater pH sensitivity.

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Fluorescent protein (FP)-based biosensors are genetically encoded tools that enable the imaging of biological processes in the context of cells, tissues, or live animals. Though widely used in biological research, practically all existing biosensors are far from ideal in terms of their performance, properties, and applicability for multiplexed imaging. These limitations have inspired researchers to explore an increasing number of innovative and creative ways to improve and maximize biosensor performance.

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