Chemical Synthesis of the Fluorescent, Cyclic Dinucleotides c(th)GAMP

The cGAS-STING pathway is known for its role in sensing cytosolic DNA introduced by a viral infection, bacterial invasion or tumorigenesis. Free DNA is recognized by the cyclic GMP-AMP synthase (cGAS) catalyzing the production of 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (2',3'-cGAMP) in mammals. This cyclic dinucleotide acts as a second messenger, activating the stimulator of interferon genes (STING) that finally triggers the transcription of interferon genes and inflammatory cytokines. Due to the therapeutic potential of this pathway, both the production and the detection of cGAMP via fluorescent moieties for assay development is of great importance. Here, we introduce the paralleled synthetic access to the intrinsically fluorescent, cyclic dinucleotides 2'3'-c(th)GAMP and 3'3'-c(th)GAMP based on phosphoramidite and phosphate chemistry, adaptable for large scale synthesis. We examine their binding properties to murine and human STING and confirm biological activity including interferon induction by 2'3'-c(th)GAMP in THP-1 monocytes. Two-photon imaging revealed successful cellular uptake of 2'3'-c(th)GAMP in THP-1 cells

A Click-Chemistry Linked 2’3’-cGAMP Analog

2’3’-cGAMP is an uncanonical cyclic dinucleotide where one A and one G base are connected via a 3’-5’ and a unique 2’-5’ linkage. The molecule is produced by the cyclase cGAS in response to cytosolic DNA binding. cGAMP activates STING and hence one of the most powerful pathways of innate immunity. cGAMP analogs with uncharged linkages that feature better cellular penetrability are currently highly desired. Here, we report the synthesis of a cGAMP analog with one amide and one triazole linkage. The molecule is best prepared via a first Cu(I) catalysed click reaction which establishes the triazole, while the cyclization is achieved by macrolactamization

STING agonism turns human T cells into interferon‐producing cells but impedes their functionality

The cGAS‐STING (cyclic GMP‐AMP synthase‐stimulator of interferon genes) axis is the predominant DNA sensing system in cells of the innate immune system. However, human T cells also express high levels of STING, while its role and physiological trigger remain largely unknown. Here, we show that the cGAS‐STING pathway is indeed functional in human primary T cells. In the presence of a TCR‐engaging signal, both cGAS and STING activation switches T cells into type I interferon‐producing cells. However, T cell function is severely compromised following STING activation, as evidenced by increased cell death, decreased proliferation, and impaired metabolism. Interestingly, these different phenotypes bifurcate at the level of STING. While antiviral immunity and cell death require the transcription factor interferon regulatory factor 3 (IRF3), decreased proliferation is mediated by STING independently of IRF3. In summary, we demonstrate that human T cells possess a functional cGAS‐STING signaling pathway that can contribute to antiviral immunity. However, regardless of its potential antiviral role, the activation of the cGAS‐STING pathway negatively affects T cell function at multiple levels. Taken together, these results could help inform the future development of cGAS‐STING‐targeted immunotherapies

Investigation of the Deprotonative Generation and Borylation of Diamine-Ligated α-Lithiated Carbamates and Benzoates by in situ IR spectroscopy

Diamine-mediated α-deprotonation of <i>O</i>-alkyl carbamates or benzoates with alkyllithium reagents, trapping of the carbanion with organoboron compounds, and 1,2-metalate rearrangement of the resulting boronate complex are the primary steps by which organoboron compounds can be stereoselectively homologated. Although the final step can be easily monitored by ¹¹B NMR spectroscopy, the first two steps, which are typically carried out at cryogenic temperatures, are less well understood owing to the requirement for specialized analytical techniques. Investigation of these steps by in situ IR spectroscopy has provided invaluable data for optimizing the homologation reactions of organoboron compounds. Although the deprotonation of benzoates in noncoordinating solvents is faster than that in ethereal solvents, the deprotonation of carbamates shows the opposite trend, a difference that has its origin in the propensity of carbamates to form inactive parasitic complexes with the diamine-ligated alkyllithium reagent. Borylation of bulky diamine-ligated lithiated species in toluene is extremely slow, owing to the requirement for initial complexation of the oxygen atoms of the diol ligand on boron with the lithium ion prior to boron–lithium exchange. However, ethereal solvent, or very small amounts of THF, facilitate precomplexation through initial displacement of the bulky diamines coordinated to the lithium ion. Comparison of the carbonyl stretching frequencies of boronates derived from pinacol boronic esters with those derived from trialkylboranes suggests that the displaced lithium ion is residing on the pinacol oxygen atoms and the benzoate/carbamate carbonyl group, respectively, explaining, at least in part, the faster 1,2-metalate rearrangements of boronates derived from the trialkylboranes

Stone artefacts on the seabed at a submerged freshwater spring confirm a drowned cultural landscape in Murujuga, Western Australia

We report the discovery and identification of five ancient stone artefacts associated with a submerged freshwater spring at the underwater archaeological site WH1 in Murujuga (Dampier Archipelago), Western Australia. A limiting date applied to the site based on timing of inundation suggests it was occupied in the Late Pleistocene or Early Holocene. The site is situated well below the intertidal zone having been recorded at 14 m depth in Flying Foam Passage. This discovery highlights the high potential of these submerged springs as archaeological survey targets. We discuss results of a recent survey that expands the number of confirmed artefacts located at WH1 and the geomorphological context in a large calcareous depression associated with a freshwater source. This study demonstrates how submerged landscape research using a suite of technologies can reveal archaeological assemblages in this tropical geomorphological environment, and that adapted techniques could be applied to other tropical conditions such as mangrove coasts, large deltaic plains, or reef-building environments. There are likely thousands of drowned archaeological sites on the continental shelf of the tropics, extending from the intertidal zone to the lowest point of the culturally occupied landmass, at approximately 130 m below modern sea level

Mutations in Zebrafish lrp2 Result in Adult-Onset Ocular Pathogenesis That Models Myopia and Other Risk Factors for Glaucoma

The glaucomas comprise a genetically complex group of retinal neuropathies that typically occur late in life and are characterized by progressive pathology of the optic nerve head and degeneration of retinal ganglion cells. In addition to age and family history, other significant risk factors for glaucoma include elevated intraocular pressure (IOP) and myopia. The complexity of glaucoma has made it difficult to model in animals, but also challenging to identify responsible genes. We have used zebrafish to identify a genetically complex, recessive mutant that shows risk factors for glaucoma including adult onset severe myopia, elevated IOP, and progressive retinal ganglion cell pathology. Positional cloning and analysis of a non-complementing allele indicated that non-sense mutations in low density lipoprotein receptor-related protein 2 (lrp2) underlie the mutant phenotype. Lrp2, previously named Megalin, functions as an endocytic receptor for a wide-variety of bioactive molecules including Sonic hedgehog, Bone morphogenic protein 4, retinol-binding protein, vitamin D-binding protein, and apolipoprotein E, among others. Detailed phenotype analyses indicated that as lrp2 mutant fish age, many individuals—but not all—develop high IOP and severe myopia with obviously enlarged eye globes. This results in retinal stretch and prolonged stress to retinal ganglion cells, which ultimately show signs of pathogenesis. Our studies implicate altered Lrp2-mediated homeostasis as important for myopia and other risk factors for glaucoma in humans and establish a new genetic model for further study of phenotypes associated with this disease