Recent advances in bioluminescent probes for neurobiology.

Bioluminescence is a popular modality for imaging in living organisms. The platform relies on enzymatically (luciferase) generated light via the oxidation of small molecule luciferins. Since no external light is needed for photon production, there are no concerns with background autofluorescence or photobleaching over time-features that have historically limited other optical readouts. Bioluminescence is thus routinely used for longitudinal tracking across whole animals. Applications in the brain, though, have been more challenging due to a lack of sufficiently bioavailable, bright, and easily multiplexed probes. Recent years have seen the development of designer luciferase and luciferin pairs that address these issues, providing more sensitive and real-time readouts of biochemical features relevant to neurobiology. This review highlights many of the advances in bioluminescent probe design, with a focus on the small molecule light emitter, the luciferin. Specific efforts to improve luciferin pharmacokinetics and tissue-penetrant emission are covered, in addition to applications that such probes have enabled. The continued development of improved bioluminescent probes will aid in illuminating critical neurochemical processes in the brain.

Expedient Synthesis and Characterization of π-Extended Luciferins.

Bioluminescence imaging enables the sensitive tracking of cell populations and the visualization of biological processes in living systems. Bioluminescent luciferase/luciferin pairs with far-red and near-infrared emission benefit from the reduced competitive absorption by blood and tissue while also facilitating multiplexing strategies. Luciferins with extended π-systems, such as AkaLumine and recently reported CouLuc-1 and -3, can be used for bioluminescence imaging in this long wavelength regime. Existing synthetic routes to AkaLumine and similar π-extended compounds require a multistep sequence to install the thiazoline heterocycle. Here we detail the development of a two-step strategy for accessing these molecules via a Horner-Wadsworth-Emmons reaction and cysteine condensation sequence from readily available aldehyde starting materials. We detail an improved synthesis of AkaLumine, as well as the corresponding two-carbon homologues, Tri- and Tetra-AkaLumine. We then extended this approach to prepare coumarin- and naphthalene-derived luciferins. These putative luciferins were tested against a panel of luciferases to identify capable emitters. Of these, an easily prepared naphthalene derivative exhibits photon emission on par with that of the broadly used Akaluc/AkaLumine pair with similar emission maxima. Overall, this chemistry provides efficient access to several bioluminescent probes for a variety of imaging applications.

Red-Shifted Coumarin Luciferins for Improved Bioluminescence Imaging.

Multicomponent bioluminescence imaging in vivo requires an expanded collection of tissue-penetrant probes. Toward this end, we generated a new class of near-infrared (NIR) emitting coumarin luciferin analogues (CouLuc-3s). The scaffolds were easily accessed from commercially available dyes. Complementary mutant luciferases for the CouLuc-3 analogues were also identified. The brightest probes enabled sensitive imaging in vivo. The CouLuc-3 scaffolds are also orthogonal to popular bioluminescent reporters and can be used for multicomponent imaging applications. Collectively, this work showcases a new set of bioluminescent tools that can be readily implemented for multiplexed imaging in a variety of biological settings.

Enhancing Alkyne-Based Raman Tags with a Sulfur Linker.

Alkyne-based Raman tags have proven their utility for biological imaging. Although the alkynyl stretching mode is a relatively strong Raman scatterer, the detection sensitivity of alkyne-tagged compounds is ultimately limited by the magnitude of the probe's Raman response. In order to improve the performance of alkyne-based Raman probes, we have designed several tags that benefit from π-π conjugation as well as from additional n-π conjugation with a sulfur linker. We show that the sulfur linker provides additional enhancement and line width narrowing, offering a simple yet effective strategy for improving alkyne-based Raman tags. We validate the utility of various sulfur-linked alkyne tags for cellular imaging through stimulated Raman scattering microscopy.

Carboxymethylcellulose excipient allergy: a case report.

BACKGROUND: Excipients are widely used in pharmaceuticals, detergents, food, and drink because of their properties of low toxicity and hypoallergenicity. The excipient carboxymethylcellulose is used extensively as a thickener in foods such as baked goods, ice cream, gluten free, and reduced fat products, where it may be labeled as e-number E466. However, excipients can rarely cause type 1 hypersensitivity reactions. Several publications have described systemic allergy following carboxymethylcellulose exposure in pharmaceuticals, particularly systemic corticosteroids. Furthermore, there is one reported case in the literature of anaphylaxis following food containing carboxymethylcellulose. CASE PRESENTATION: We identify a case of anaphylaxis in a 45-year-old atopic Caucasian woman on receiving an injectable suspension of the corticosteroid triamcinolone acetonide containing carboxymethylcellulose, and subsequent allergic symptoms on reexposure to carboxymethylcellulose in a commercial drink. Diagnosis of carboxymethylcellulose excipient allergy was confirmed through skin prick testing using Celluvisc carmellose 0.5% eye drops, which contain carboxymethylcellulose as the active ingredient. CONCLUSION: This case highlights the importance of identifying excipients such as carboxymethylcellulose as causes of allergy, to reduce burden of further hypersensitivity reactions, not just to drugs but to other consumables.

Photoredox/Nickel Dual Catalytic Cross-Coupling of Potassium Thiomethyltrifluoroborates with Aryl and Heteroaryl Bromides.

The cross-coupling of S-aryl and S-alkyl potassium thiomethyltrifluoroborates with aryl and heteroaryl bromides is reported via photoredox/nickel dual catalysis. The transformation is achieved under mild conditions with commercially available or readily prepared, air stable reagents and affords benzylthioether products in moderate to good yields with good functional group tolerance. A practical and improved synthesis of potassium thiomethyltrifluoroborates is also reported that affords access to previously undescribed reagents.

Immunotherapy in gastrointestinal cancer: The current scenario and future perspectives.

Gastrointestinal cancers include colorectal, gastric, oesophageal, pancreatic and liver cancers. They continue to be a significant cause of mortality and morbidity worldwide. Current treatment strategies include chemotherapy, surgery, radiotherapy and targeted therapies. Immunotherapy has recently been incorporated in treatment regimens for some gastrointestinal malignancies and research into different immune modifying treatments is being carried out in this context. Approaches to immune modulation such as vaccination, adoptive cell therapy and checkpoint inhibition have shown varying clinical benefit, with most of the benefit seen in checkpoint inhibition. This review summarises recent advances and future direction of immunotherapy in patients with gastrointestinal malignancies.

Efficient Synthesis of All-Carbon Quaternary Centers via the Conjugate Addition of Functionalized Monoorganozinc Bromides.

The conjugate addition of organometallic reagents to α,ß-unsaturated carbonyls represents an important method to generate C-C bonds in the preparation of all-carbon quaternary centers. Though conjugate additions of organometallic reagents are typically performed utilizing highly reactive organolithium or Grignard reagents, organozinc reagents have garnered attention for their enhanced chemoselectivity and mild reactivity. Despite numerous recent advances with more reactive diorganozinc and mixed diorganozinc reagents, the generation of all-carbon quaternary centers via the conjugate addition of functionalized monoorganozinc reagents remains a challenge. This protocol details a convenient and mild "one-pot" preparation and copper mediated conjugate addition of functionalized monoorganozinc bromides to cyclic α,ß-unsaturated carbonyls to afford a broad scope of all-carbon quaternary centers in generally excellent yield and diastereoselectivity. Key to the development of this technology is the utilization of DMA as a reaction solvent with TMSCl as a Lewis acid. Notable advantages to this methodology include the operational simplicity of the organozinc reagent preparation afforded by the utilization of DMA as a solvent, as well as an efficient conjugate addition mediated by various Cu(I) and Cu(II) salts. Moreover, an intermediate silyl enol ether can be isolated utilizing a modified workup procedure. The substrate scope is limited to cyclic unsaturated ketones, and the conjugate addition is impeded by stabilized (e.g., allyl, enolate, homoenolate) and sterically encumbered (e.g., neopentyl, o-aryl) monoorganozinc reagents. Conjugate additions to five- and seven-membered rings were effective, albeit in lower yields compared with six-membered ring substrates.

Novel Burkholderia mallei virulence factors linked to specific host-pathogen protein interactions.

Burkholderia mallei is an infectious intracellular pathogen whose virulence and resistance to antibiotics makes it a potential bioterrorism agent. Given its genetic origin as a commensal soil organism, it is equipped with an extensive and varied set of adapted mechanisms to cope with and modulate host-cell environments. One essential virulence mechanism constitutes the specialized secretion systems that are designed to penetrate host-cell membranes and insert pathogen proteins directly into the host cell's cytosol. However, the secretion systems' proteins and, in particular, their host targets are largely uncharacterized. Here, we used a combined in silico, in vitro, and in vivo approach to identify B. mallei proteins required for pathogenicity. We used bioinformatics tools, including orthology detection and ab initio predictions of secretion system proteins, as well as published experimental Burkholderia data to initially select a small number of proteins as putative virulence factors. We then used yeast two-hybrid assays against normalized whole human and whole murine proteome libraries to detect and identify interactions among each of these bacterial proteins and host proteins. Analysis of such interactions provided both verification of known virulence factors and identification of three new putative virulence proteins. We successfully created insertion mutants for each of these three proteins using the virulent B. mallei ATCC 23344 strain. We exposed BALB/c mice to mutant strains and the wild-type strain in an aerosol challenge model using lethal B. mallei doses. In each set of experiments, mice exposed to mutant strains survived for the 21-day duration of the experiment, whereas mice exposed to the wild-type strain rapidly died. Given their in vivo role in pathogenicity, and based on the yeast two-hybrid interaction data, these results point to the importance of these pathogen proteins in modulating host ubiquitination pathways, phagosomal escape, and actin-cytoskeleton rearrangement processes.