Despite recent advances in crystallography of G protein-coupled receptors (GPCRs), little is known about the mechanism of their activation process, as only the β2 adrenergic receptor (β2AR) and rhodopsin have been crystallized in fully active conformations. Here, we report the structure of an agonist-bound, active state of the human M2 muscarinic acetylcholine receptor stabilized by a G-protein mimetic camelid antibody fragment isolated by conformational selection using yeast surface display. In addition to the expected changes in the intracellular surface, the structure reveals larger conformational changes in the extracellular region and orthosteric binding site than observed in the active states of the β2AR and rhodopsin. We also report the structure of the M2 receptor simultaneously binding the orthosteric agonist iperoxo and the positive allosteric modulator LY2119620. This structure reveals that LY2119620 recognizes a largely pre-formed binding site in the extracellular vestibule of the iperoxo-bound receptor, inducing a slight contraction of this outer binding pocket. These structures offer important insights into activation mechanism and allosteric modulation of muscarinic receptors.

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Growth inhibition and apoptotic/necrotic phenotype was observed in nanogold particle (AuNP)-treated human chronic myelogenous leukemia cells. To elucidate the underlying cellular mechanisms, proteomic techniques including two-dimensional electrophoresis/mass spectrometry and protein microarrays were utilized to study the differentially expressed proteome and phosphoproteome, respectively. Systems biology analysis of the proteomic data revealed that unfolded protein-associated endoplasmic reticulum (ER) stress response was the predominant event. Concomitant with transcriptomic analysis using mRNA expression, microarrays show ER stress response in the AuNP-treated cells. The ER stress protein markers' expression assay unveiled AuNPs as an efficient cellular ER stress elicitor. Upon ER stress, cellular responses, including reactive oxygen species increase, mitochondrial cytochrome c release, and mitochondria damage, chronologically occurred in the AuNP-treated cells. Conclusively, this study demonstrates that AuNPs cause cell death through induction of unmanageable ER stress.

We studied the chemical processes that take place during hydrothermal gelation of graphene oxide (GO), quantifying the reaction products generated during hydrothermal reduction. The gelation proceeds with disproportionation of GO yielding a large amount of CO2 (about a quarter of the original mass of GO), organic acidic fragments, and CO. The CO2 that is formed is trapped in the hydrogel creating macroscopic voids which can lead to cracking of the hydrogel during compression. We were able to quantify the amount of CO2 produced in situ by adding ammonia during the synthesis, and converting CO2 into ionic carbonate species that we could easily quantify by titration. We used titration to evaluate the formation of organic acidic fragments too and evaluated the amount of H2O and CO produced by thermogravimetric analysis and mass balance. The conversion of CO2 into ionic species allowed us to produce void-free hydrogels which remain structurally stable after extensive compression. However, such hydrogels on average showed lower mechanical strength and electrical conductivity than the hydrogels with voids. This is a result of the difference in chemistry and morphology between hydrogels reduced under acidic pH and basic pH. Our work provides for the first time a clear quantitative estimate of CO2 evolution and organic fragment formation during hydrothermal reduction of GO, an overall picture of the reaction products, and a deepened understanding of the conditions that can be used to prepare stronger and more conductive graphene hydrogels and aerogels.