NRAS Mutant Dictates AHCYL1-Governed ER Calcium Homeostasis for Melanoma Tumor Growth.

Calcium homeostasis is critical for cell proliferation, and emerging evidence shows that cancer cells exhibit altered calcium signals to fulfill their need for proliferation. However, it remains unclear whether there are oncogene-specific calcium homeostasis regulations that can expose novel therapeutic targets. Here, from RNAi screen, we report that adenosylhomocysteinase like protein 1 (AHCYL1), a suppressor of the endoplasmic reticulum (ER) calcium channel protein inositol trisphosphate receptor (IP3R), is selectively upregulated and critical for cell proliferation and tumor growth potential of human NRAS-mutated melanoma, but not for melanoma expressing BRAF V600E. Mechanistically, AHCYL1 deficiency results in decreased ER calcium levels, activates the unfolded protein response (UPR), and triggers downstream apoptosis. In addition, we show that AHCYL1 transcription is regulated by activating transcription factor 2 (ATF2) in NRAS-mutated melanoma. Our work provides evidence for oncogene-specific calcium regulations and suggests AHCYL1 as a novel therapeutic target for RAS mutant-expressing human cancers, including melanoma. IMPLICATIONS: Our findings suggest that targeting the AHCYL1-IP3R axis presents a novel therapeutic approach for NRAS-mutated melanomas, with potential applicability to all cancers harboring RAS mutations, such as KRAS-mutated human colorectal cancers.

Easily scalable multi-color DMD-based structured illumination microscopy.

Structured illumination microscopy (SIM) achieves super-resolution imaging using a series of phase-shifted sinusoidal illumination patterns to down-modulate high spatial-frequency information of samples. Digital micromirror devices (DMDs) have been increasingly used to generate SIM illumination patterns due to their high speed and moderate cost. However, a DMD micromirror array's blazed grating structure causes strong angular dispersion for different wavelengths of light, thus severely hampering its application in multicolor imaging. We developed a multi-color DMD-SIM setup that employs a diffraction grating to compensate the DMD's dispersion and demonstrate super-resolution SIM imaging of both fluorescent beads and live cells samples with four color channels. This simple but effective approach can be readily scaled to more color channels, thereby greatly expanding the application of SIM in the study of complex multi-component structures and dynamics in soft matter systems.

Ornithinibacillus caprae sp. nov., a moderate halophile isolated from the hides of a white goat.

A novel Gram-stain-positive, motile, moderate halophile, strain L9T, was isolated from hides of white goat in China. The isolate grew optimally at 30 °C, at pH 7 and with 5-10% (w/v) NaCl. The predominant menaquinone was MK-7, and the major cellular fatty acids were identified as iso-C15:0 and anteiso-C15:0. The peptidoglycan amino acid type was determined to be A4ß, containing L-ornithine and D-aspartic as diagnostic amino acids. The phospholipids were dominated by diphosphatidylglycerol, phosphatidylglycerol, one unidentified aminophospholipid and two unidentified phospholipids. Genome sequencing resulted in a genome size of 4.0 Mbp and a DNA G + C content of 35.9 mol%. Phylogenetic trees based on the 16S rRNA gene sequences showed the isolate to be closely related to Oceanobacillus limi H9BT (98.2% similarity) and Ornithinibacillus halophilus G8BT (97.5% similarity). The ANI and dDDH values between strain L9T and the closely related species were 69.8-76.1% and 13.0-20.5%, respectively. On the basis of the data presented, strain L9T represents a novel species of the genus Ornithinibacillus, for which the name Ornithinibacillus caprae sp. nov. is proposed. The type strain is L9T (= KCTC 43176T = CGMCC 1.17659T).

TCR-pMHC bond conformation controls TCR ligand discrimination.

A major unanswered question is how a TCR discriminates between foreign and self-peptides presented on the APC surface. Here, we used in situ fluorescence resonance energy transfer (FRET) to measure the distances of single TCR-pMHC bonds and the conformations of individual TCR-CD3ζ receptors at the membranes of live primary T cells. We found that a TCR discriminates between closely related peptides by forming single TCR-pMHC bonds with different conformations, and the most potent pMHC forms the shortest bond. The bond conformation is an intrinsic property that is independent of the binding affinity and kinetics, TCR microcluster formation, and CD4 binding. The bond conformation dictates the degree of CD3ζ dissociation from the inner leaflet of the plasma membrane via a positive calcium signaling feedback loop to precisely control the accessibility of CD3ζ ITAMs for phosphorylation. Our data revealed the mechanism by which a TCR deciphers the structural differences among peptides via the TCR-pMHC bond conformation.

Development and Characterization of an Endotoxemia Model in Zebra Fish.

Endotoxemia is a condition in which endotoxins enter the blood stream and cause systemic and sometimes lethal inflammation. Zebra fish provides a genetically tractable model organism for studying innate immunity, with additional advantages in live imaging and drug discovery. However, a bona fide endotoxemia model has not been established in zebra fish. Here, we have developed an acute endotoxemia model in zebra fish by injecting a single dose of LPS directly into the circulation. Hallmarks of human acute endotoxemia, including systemic inflammation, extensive tissue damage, circulation blockade, immune cell mobilization, and emergency hematopoiesis, were recapitulated in this model. Knocking out the adaptor protein Myd88 inhibited systemic inflammation and improved zebra fish survival. In addition, similar alternations of pathways with human acute endotoxemia were detected using global proteomic profiling and MetaCore™ pathway enrichment analysis. Furthermore, treating zebra fish with a protein tyrosine phosphatase nonreceptor type 11 (Shp2) inhibitor decreased systemic inflammation, immune mobilization, tissue damage, and improved survival in the endotoxemia model. Together, we have established and characterized the phenotypic and gene expression changes of a zebra fish endotoxemia model, which is amenable to genetic and pharmacological discoveries that can ultimately lead to a better mechanistic understanding of the dynamics and interplay of the innate immune system.