A neurodevelopmental disorder mutation locks G proteins in the transitory pre-activated state.

Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. GαoK46E has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein ßγ subunits. In cells with physiological concentrations of nucleotide, GαoK46E forms a stable complex with receptors and Gßγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gαo, without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells.

Primary cervicothoracic melanoma of spinal cord: a case report and literature review.

A 53-year-old male patient presented progressive numbness and weakness in the right limbs for a 2-year duration. Magnetic resonance imaging scans revealed an intramedullary lesion crossed over cervical and thoracic levels accompanied by syringomyelia at the proximal end of the lesion. The patient underwent subtotal resection of the neoplasm. The histological findings of the tumor were consistent with primary intramedullary malignant melanoma and not initial ependymoma after careful dermatologic and ophthalmologic re-examination. Primary melanoma of the spinal cord, particularly cervicothoracic localization with syringomyelia, is seldom reported in the literature. We report a case of this uncommon tumor and also discuss the clinical course, diagnosis, and treatment.

Stabilization of interdomain interactions in G protein α subunits as a determinant of Gαi subtype signaling specificity.

Highly homologous members of the Gαi family, Gαi1-3, have distinct tissue distributions and physiological functions, yet their biochemical and functional properties are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gαi1 effector that is poorly activated by Gαi2. In a proteomic proximity labeling screen we observed a strong preference for Gαi1 relative to Gαi2 with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gαi1 into Gαi2 or substitution of a single amino acid, A230 in Gαi2 with the corresponding D in Gαi1, largely rescues PRG activation and interactions with other potential Gαi targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, separation at the HD-Ras-like domain (RLD) interface is more pronounced in Gαi2 than Gαi1. Mutation of A230 to D in Gαi2 stabilizes HD-RLD interactions via ionic interactions with R145 in the HD which in turn modify the conformation of Switch III. These data support a model where D229 in Gαi1 interacts with R144 and stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gαi2 is unable to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gαi1 or Gαi2 by G protein-coupled receptors.

Molecular annotation of G protein variants in a neurological disorder.

Heterotrimeric G proteins transduce extracellular chemical messages to generate appropriate intracellular responses. Point mutations in GNAO1, encoding the G protein αo subunit, have been implicated in a pathogenic condition characterized by seizures, movement disorders, intellectual disability, and developmental delay (GNAO1 disorder). However, the effects of these mutations on G protein structure and function are unclear. Here, we report the effects of 55 mutations on Gαo conformation, thermostability, nucleotide binding, and hydrolysis, as well as interaction with Gßγ subunits, receptors, and effectors. Our effort reveals four functionally distinct groups of mutants, including one group that sequesters receptors and another that sequesters Gßγ, both acting in a genetically dominant manner. These findings provide a more comprehensive understanding of disease-relevant mutations and reveal that GNAO1 disorder is likely composed of multiple mechanistically distinct disorders that will likely require multiple therapeutic strategies.

Stabilization of Interdomain Interactions in G protein αi Subunits Determines Gαi Subtype Signaling Specificity.

Highly homologous members of the Gαi family, Gαi1-3, have distinct tissue distributions and physiological functions, yet the functional properties of these proteins with respect to GDP/GTP binding and regulation of adenylate cyclase are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gαi1 effector, however, it is poorly activated by Gαi2. Here, in a proteomic proximity labeling screen we observed a strong preference for Gαi1 relative to Gαi2 with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gαi1 into Gαi2 or substitution of a single amino acid, A230 in Gαi2 to the corresponding D in Gαi1, largely rescues PRG activation and interactions with other Gαi targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, dynamic separation at the HD-Ras-like domain (RLD) interface is prevalent in Gαi2 relative to Gαi1 and that mutation of A230s4h3.3 to D in Gαi2 stabilizes HD-RLD interactions through formation of an ionic interaction with R145HD.11 in the HD. These interactions in turn modify the conformation of Switch III. These data support a model where D229s4h3.3 in Gαi1 interacts with R144HD.11 stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gαi2 is unable to form the "ionic lock" to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gαi1 or Gαi2 by GPCRs.

Heterogeneous porous PLLA/PCL fibrous scaffold for bone tissue regeneration.

Bone tissue engineering has become one of the most promising therapeutic methods to treat bone defects. A suitable scaffolding material to regenerate new bone tissues should have a high specific surface area, high porosity and a suitable surface structure which benefit cell attachment, proliferation, and differentiation. In this study, an acetone post-treatment strategy was developed to generate heterogeneous structure. After PLLA/PCL nanofibrous membranes were electrospun and collected, they were treated with acetone to generate a highly porous structure. Meanwhile, part of PCL was extracted from the fibre and enriched on the fibre surface. The cell affinity of the nanofibrous membrane was verified by human osteoblast-like cells assay. The proliferation rate of heterogeneous samples increased 190.4 %, 265.5 % and 137.9 % at day 10 compared with pristine samples. These results demonstrated that the heterogeneous PLLA/PCL nanofibrous membranes could enhance osteoblast adhesion and proliferation. With high surface area (average surface area 36.302 m2/g) and good mechanical properties (average Young's modulus 1.65 GPa and average tensile strength 5.1 MPa), the heterogeneous PLLA/PCL membrane should have potential applications in the field of bone regeneration.

Tunable Graphene/Nitrocellulose Temperature Alarm Sensors.

Tunable temperature alarm sensors were prepared using multilayer graphene and nitrocellulose (NC) to reliably monitor early high temperature risks. The graphene/NC alarm sensor keeps in a state of electrical insulation, however, turns electrically conductive at high temperatures, such as encountering a flame attack. Its response time is limited to only a few seconds because of a quick chemical reaction of NC. The 90% graphene/NC (wt % ratio 1:9) composite alarm sensor stably remains insulated at an ambient temperature of 200 °C, resulting in a satisfactory responsive temperature (232 °C), instant response time (4.4 s), and sustained working time in the flame below the ignition temperature of most combustibles. Furthermore, the response temperature and time of the alarm sensor can be tuned by graphene/NC ratios to reduce the fire risk of various combustible materials in different fire-prone scenarios and thus has promising applications in both indoor and outdoor environments. The sensor has also proven to work in the form of paint, wallpaper, and other composites due to its superior flame retardancy property, as well as under extreme conditions (i.e., underwater and vacuum).

Rare Cervicothoracic Intramedullary and Extramedullary Lipoma.

We report a rare case of cervicothoracic intramedullary and extramedullary lipoma. Complete resection of the extramedullary lipoma and almost complete resection of the intramedullary lipoma were performed using a microscope, followed by posterior fusion and internal fixation from C4-T2 to maintain the stability of the cervicothoracic junction. Despite the high risk, it was still necessary to perform the decompression surgery and the surgical results were favorable.

Hierarchical Porous Poly(l-lactic acid) Nanofibrous Membrane for Ultrafine Particulate Aerosol Filtration.

Ultrafine particulate aerosols less than 100 nm diffuse randomly in the air and are hazardous to the environment and human health. However, no technical standards or commercial products are available for filtering particle sizes under 100 nm yet. Here, we report the development of a porous poly(l-lactic acid) (PLLA) nanofibrous membrane filter with an ultra-high specific surface area via electrospinning and a post-treatment process. After PLLA fibres were electrospun and collected, they were treated by acetone to generate a blossoming porous structure throughout each individual fibre. Characterizations of morphology, crystallinity, and mechanical and thermal properties demonstrated that the porous structure can be attributed to the nonsolvent-induced spinodal phase separation during electrospinning and solvent-induced recrystallization during post treatment. The blossoming porous structure with high specific surface area contributed to excellent filtration efficiency (99.99%) for sodium chloride (NaCl) ultrafine aerosol particles (30-100 nm) with a low pressure drop (110-230 Pa). Notably, under 7.8 cm/s air flow rate, the membrane samples performed better for filtering smaller-sized aerosol particles than the larger ones when evaluated by the quality factor (0.07). Finally, this finding demonstrates that the electrospun membrane with a hierarchical pore structure and high specific surface area hold great potential in applications as air-filtering materials.

Genome Sequences of Cluster K Mycobacteriophages Deby, LaterM, LilPharaoh, Paola, SgtBeansprout, and Sulley.

Mycobacteriophages Deby, LaterM, LilPharaoh, Paola, SgtBeansprout, and Sulley were isolated from soil using Mycobacterium smegmatis mc2155. Genomic analysis indicated that they belong to subclusters K1 and K5. Their genomic architectures are typical of cluster K mycobacteriophages, with most variability occurring on the right end of the genome sequence.