The photoreceptor cyclic nucleotide-gated (CNG) channel plays a pivotal role in phototransduction and cellular calcium homeostasis. Mutations in the cone photoreceptor CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. CNG channel deficiency leads to endoplasmic reticulum (ER) stress-associated cone apoptosis, protein mislocalization, and ER calcium dysregulation. This work investigated the potential mechanisms of protein mislocalization associated with ER calcium dysregulation using Cnga3-/- mice lacking ER Ca2+ channel ryanodine receptor 2 (RyR2) specifically in cones. Deletion of Ryr2 improved outer segment (OS) localization of the cone proteins M-opsin, S-opsin, and cone phosphodiesterase subunit α' (PDE6C) and decreased inner segment localization. One-month-old Cnga3-/- mice showed ∼30% of M-opsin, 55% of S-opsin, and 50% of PDE6C localized to the OS. Cnga3-/- mice with Ryr2 deletion at the same age showed almost 60% of M-opsin, 70% of S-opsin, and 70% of PDE6C localized to the OS. Deletion of Ryr2 nearly completely reversed elevations of the ER stress markers phospho-IRE1α and phospho-eIF2α and suppressed cone apoptosis. Consistent with the improved cone protein localization and reduced ER stress/cone apoptosis, cone survival was improved by deletion of Ryr2 The number of cones was increased by ∼28% in 2- to 4-month-old Cnga3-/- mice with Ryr2 deletion compared with age-matched Cnga3-/- mice. This work demonstrates a role of RyR2/ER calcium dysregulation in protein mislocalization, ER stress, and cone death. The findings provide novel insights into the mechanisms of photoreceptor degeneration and support strategies targeting ER calcium regulation to manage retinal degeneration.

Three new cyclic peptides including a cyclic tetrapeptide (1), an aspochracin-type cyclic tripeptide sclerotiotide L (2) and a diketopiperazine dimer (3), have been isolated from the ethyl acetate extract of a marine sponge-derived fungus Aspergillus violaceofuscus. The structures of all compounds were unambiguously elucidated on the basis of HRESIMS, 1D and 2D NMR spectroscopic data, MS/MS experiments and chemical methods. Compounds 1 and 3 showed anti-inflammatory activity against IL-10 expression of the LPS-induced THP-1 cells with inhibitory rates of 84.3 and 78.1% respectively at concentration of 10 μM.

Oral squamous cancers (OSC) are hallmarked by poor prognosis, delayed clinical detection, and a lack of defined, characteristic biomarkers. By screening combinatorial one-bead one-compound (OBOC) peptide libraries against oral squamous cancer cell lines, two cyclic peptide ligands, LLY12 and LLY13 were previously identified. These ligands are capable of specific binding to the oral cancer cell lines (MOK-101, HSC-3, SCC-4 and SCC-10a) but not non-cancerous keratinocytes, leukocytes, fibroblast, and endothelial cells. These two peptides were synthesized and evaluated for their binding property, cytotoxicity and cell permeability. In vitro studies indicate that both LLY12 and LLY13 were able to bind to oral cancer cells with high specificity but did not show any cytotoxicity against human keratinocytes. Biotinylated LLY13, in complex with streptavidin-alexa488 was taken up by live oral cancer cells, thus rendering it as an excellent candidate vehicle for efficient delivery of drug loaded-nanoparticles. In vivo and ex vivo near infra-red fluorescence imaging studies confirmed the in vivo targeting efficiency and specificity of LLY13 in oral cancer orthotopic murine xenograft model. In vivo studies also showed that LLY13 was able to accumulate in the OSC tumors and demarcate the tumor margins in orthotopic xenograft model. Together, our data supports LLY13 as a promising theranostic agent against OSC.

A key question that has remained unanswered is how pathogenic fungi switch from vegetative growth to infection-related morphogenesis during a disease cycle. Here, we identify a fungal oxylipin analogous to the phytohormone jasmonic acid (JA), as the principal regulator of such a developmental switch to isotropic growth and pathogenicity in the rice-blast fungus Magnaporthe oryzae. Using specific inhibitors and mutant analyses, we determined the molecular function of intrinsic jasmonates during M. oryzae pathogenesis. Loss of 12-Oxo-phytodienoic Acid (OPDA) Reductase and/or consequent reduction of jasmonate biosynthesis, prolonged germ tube growth and caused delayed initiation and improper development of infection structures in M. oryzae, reminiscent of phenotypic defects upon impaired cyclic AMP (cAMP) signaling. Chemical- or genetic-complementation completely restored proper vegetative growth and appressoria in opr1Δ. Mass spectrometry-based quantification revealed increased OPDA accumulation and significantly decreased jasmonate levels in opr1Δ. Most interestingly, exogenous jasmonate restored proper appressorium formation in pth11Δ that lacks G protein/cAMP signaling; but failed to do so in the Mitogen-activated protein (MAP) kinase mutants. Epistasis analysis placed jasmonate upstream of the cAMP pathway in rice blast. Mechanistically, intrinsic jasmonate orchestrates timely cessation of the vegetative phase and induces pathogenic development via a complex regulatory interaction with the cAMP-PKA cascade and redox signaling in rice blast.

Mutations in the CNGA3 gene, which encodes the A subunit of the cyclic guanosine monophosphate (cGMP)-gated cation channel in cone photoreceptor outer segments, cause total colour blindness, also referred to as achromatopsia. Cones lacking this channel protein are non-functional, accumulate high levels of the second messenger cGMP and degenerate over time after induction of ER stress. The cell death mechanisms that lead to loss of affected cones are only partially understood. Here, we explored the disease mechanisms in the Cnga3 knockout (KO) mouse model of achromatopsia. We found that another important effector of cGMP, the cGMP-dependent protein kinase 2 (Prkg2) is crucially involved in cGMP cytotoxicity of cones in Cnga3 KO mice. Virus-mediated knockdown or genetic ablation of Prkg2 in Cnga3 KO mice counteracted degeneration and preserved the number of cones. Analysis of markers of endoplasmic reticulum stress and unfolded protein response confirmed that induction of these processes in Cnga3 KO cones also depends on Prkg2. In conclusion, we identified Prkg2 as a novel key mediator of cone photoreceptor degeneration in achromatopsia. Our data suggest that this cGMP mediator could be a novel pharmacological target for future neuroprotective therapies.

Cyclic GMP-AMP synthase (cGAS) plays a key role in the innate immune responses to both DNA and RNA virus infection. Here, we found that enterovirus 71 (EV-A71), Seneca Valley virus (SVV), and foot-and-mouth disease virus (FMDV) infection triggered mitochondria damage and mitochondrial DNA (mtDNA) release in vitro and vivo. These responses were mediated by picornavirus 2B proteins which induced mtDNA release during viral replication. SVV infection caused the opening of mitochondrial permeability transition pore (mPTP) and led to voltage-dependent anion channel 1 (VDAC1)- and BCL2 antagonist/killer 1 (Bak) and Bak/BCL2-associated X (Bax)-dependent mtDNA leakage into the cytoplasm, while EV-A71 and FMDV infection induced mPTP opening and resulted in VDAC1-dependent mtDNA release. The released mtDNA bound to cGAS and activated cGAS-mediated antiviral immune response. cGAS was essential for inhibiting EV-A71, SVV, and FMDV replication by regulation of IFN-β production. cGAS deficiency contributed to higher mortality of EV-A71- or FMDV-infected mice. In addition, we found that SVV 2C protein was responsible for decreasing cGAS expression through the autophagy pathway. The 9th and 153rd amino acid sites in 2C were critical for induction of cGAS degradation. Furthermore, we also show that EV-A71, CA16, and EMCV 2C antagonize the cGAS-stimulator of interferon genes (STING) pathway through interaction with STING, and highly conserved amino acids Y155 and S156 were critical for this inhibitory effect. In conclusion, these data reveal novel mechanisms of picornaviruses to block the antiviral effect mediated by the cGAS-STING signaling pathway, which will provide insights for developing antiviral strategies against picornaviruses.