Development of “Plug and Play” Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM

“Universal” synthetic antibody (sAB)-based fiducial marks have been generated by customized phage display selections to facilitate the rapid structure determination of G protein-coupled receptor (GPCR) signaling complexes by single-particle cryo-electron microscopy (SP cryo-EM). sABs were generated to the two major G protein subclasses: trimeric G_i and G_s, as well as mini-G_s, and were tested to ensure binding in the context of their cognate GPCRs. Epitope binning revealed that multiple distinct epitopes exist for each G(αβγ) protein. Several Gβγ-specific sABs, cross-reactive between trimeric G_i and G_s, were identified suggesting they could be used across all subclasses in a “plug and play” fashion. sABs were also generated to a representative of another class of GPCR signaling partner, G protein receptor kinase 1 (GRK1) and evaluated further, supporting the generalizability of the approach. EM data suggested that the subclass-specific sABs provide effective single and dual fiducials for multiple GPCR signaling complexes

Development of “Plug and Play” Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM

“Universal” synthetic antibody (sAB)-based fiducial marks have been generated by customized phage display selections to facilitate the rapid structure determination of G protein-coupled receptor (GPCR) signaling complexes by single-particle cryo-electron microscopy (SP cryo-EM). sABs were generated to the two major G protein subclasses: trimeric G_i and G_s, as well as mini-G_s, and were tested to ensure binding in the context of their cognate GPCRs. Epitope binning revealed that multiple distinct epitopes exist for each G(αβγ) protein. Several Gβγ-specific sABs, cross-reactive between trimeric G_i and G_s, were identified suggesting they could be used across all subclasses in a “plug and play” fashion. sABs were also generated to a representative of another class of GPCR signaling partner, G protein receptor kinase 1 (GRK1) and evaluated further, supporting the generalizability of the approach. EM data suggested that the subclass-specific sABs provide effective single and dual fiducials for multiple GPCR signaling complexes

Primate social cognition: uniquely primate, uniquely social, or just unique?

Primates undoubtedly have impressive abilities in perceiving, recognising, understanding and interpreting other individuals, their ranks and relationships; they learn rapidly in social situations, employ both deceptive and cooperative tactics to manipulate companions, and distinguish others’ knowledge from ignorance. Some evidence suggests that great apes recognize the cognitive basis of manipulative tactics and have a deeper appreciation of intention and cooperation than monkeys; and only great apes among primates show any understanding of the concept of self. None of these abilities is unique to primates, however. We distinguish (1) a package of quantitative advantages in social sophistication, evident in several broad mammalian taxa, in which neocortical enlargement is associated with social group size; from (2) a qualitative difference in understanding found in several distantly related but large-brained species, including great apes, some corvids, and perhaps elephants, dolphins, and domestic dogs. Convergence of similar abilities in widely divergent taxa should enable their cognitive basis and evolutionary origins to be determined. Cortical enlargement seems to have been evolutionarily selected by social challenges, although it confers intellectual benefits in other domains also; most likely the mechanism is more efficient memory. The taxonomic distribution of qualitatively special social skills does not point to an evolutionary origin in social challenges, and may be more closely linked to a need to acquire novel ways of dealing with the physical world; but at present research on this question remains in its infancy. In the case of great apes, their ability to learn new manual routines by parsing action components may also account for their qualitatively different social skills, suggesting that any strict partition of physical and social cognition is likely to be misleading

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

The Short-Term Effect of Weight Loss Surgery on Volumetric Breast Density and Fibroglandular Volume

Purpose: Obesity and breast density are both associated with an increased risk of breast cancer and are potentially modifiable. Weight loss surgery (WLS) causes a significant reduction in the amount of body fat and a decrease in breast cancer risk. The effect of WLS on breast density and its components has not been documented. Here, we analyze the impact of WLS on volumetric breast density (VBD) and on each of its components (fibroglandular volume and breast volume) by using three-dimensional methods. Materials and Methods: Fibroglandular volume, breast volume, and their ratio, the VBD, were calculated from mammograms before and after WLS by using Volpara™ automated software. Results: For the 80 women included, average body mass index decreased from 46.0 ± 7.22 to 33.7 ± 7.06 kg/m2. Mammograms were performed on average 11.6 ± 9.4 months before and 10.1 ± 7 months after WLS. There was a significant reduction in average breast volume (39.4 % decrease) and average fibroglandular volume (15.5 % decrease), and thus, the average VBD increased from 5.15 to 7.87 % (p < 1 × 10−9) after WLS. When stratified by menopausal status and diabetic status, VBD increased significantly in all groups but only perimenopausal and postmenopausal women and non-diabetics experienced a significant reduction in fibroglandular volume. Conclusions: Breast volume and fibroglandular volume decreased, and VBD increased following WLS, with the most significant change observed in postmenopausal women and non-diabetics. Further studies are warranted to determine how physical and biological alterations in breast density components after WLS may impact breast cancer risk.ECU Open Access Publishing Support Fun

The Advantage of Standing Up to Fight and the Evolution of Habitual Bipedalism in Hominins

BACKGROUND: Many quadrupedal species stand bipedally on their hindlimbs to fight. This posture may provide a performance advantage by allowing the forelimbs to strike an opponent with the range of motion that is intrinsic to high-speed running, jumping, rapid braking and turning; the range of motion over which peak force and power can be produced. METHODOLOGY/PRINCIPAL FINDINGS: To test the hypothesis that bipedal (i.e., orthograde) posture provides a performance advantage when striking with the forelimbs, I measured the force and energy produced when human subjects struck from "quadrupedal" (i.e., pronograde) and bipedal postures. Downward and upward directed striking energy was measured with a custom designed pendulum transducer. Side and forward strikes were measured with a punching bag instrumented with an accelerometer. When subjects struck downward from a bipedal posture the work was 43.70±12.59% (mean ± S.E.) greater than when they struck from a quadrupedal posture. Similarly, 47.49±17.95% more work was produced when subjects struck upward from a bipedal stance compared to a quadrupedal stance. Importantly, subjects did 229.69±44.19% more work in downward than upward directed strikes. During side and forward strikes the force impulses were 30.12±3.68 and 43.04±9.00% greater from a bipedal posture than a quadrupedal posture, respectively. CONCLUSIONS/SIGNIFICANCE: These results indicate that bipedal posture does provide a performance advantage for striking with the forelimbs. The mating systems of great apes are characterized by intense male-male competition in which conflict is resolved through force or the threat of force. Great apes often fight from bipedal posture, striking with both the fore- and hindlimbs. These observations, plus the findings of this study, suggest that sexual selection contributed to the evolution of habitual bipedalism in hominins

Capturing the sounds of an urban greenspace

Acoustic data can be a source of important information about events and the environment in modern cities. To date, much of the focus has been on monitoring noise pollution, but the urban soundscape contains a rich variety of signals about both human and natural phenomena. We describe the CitySounds project, which has installed enclosed sensor kits at several locations across a heavily used urban greenspace in the city of Edinburgh. The acoustic monitoring components regularly capture short clips in real-time of both ultrasonic and audible noises, for example encompassing bats, birds and other wildlife, traffic, and human. The sounds are complemented by collecting other data from sensors, such as temperature and relative humidity. To ensure privacy and compliance with relevant legislation, robust methods render completely unintelligible any traces of voice or conversation that may incidentally be overheard by the sensors. We have adopted a variety of methods to encourage community engagement with the audio data and to communicate the richness of urban soundscapes to a general audience

Structural Dynamics of G Protein-Coupled Receptor Signaling

G protein-coupled receptors (GPCRs) are versatile, ubiquitously expressed seventransmembrane domain (TMD) proteins that are conserved across the animal kingdom. In response to a strikingly diverse array of extracellular ligands such hormones, neurotransmitters, odorants, short peptides, protein ligands, and photons, GPCRs initiate a variety of intracellular signaling cascades through G protein and arrestin coupling that regulate a broad array of physiological processes including vision, olfaction, taste, neurotransmission, cellular differentiation, and immune response. The ability of GPCRs to mediate this signaling is dependent upon their TMD to undergo substantial conformational change in response to ligand binding. However, while GPCR structures determined by X-ray crystallography and cryoelectron microscopy provide invaluable temporal snapshots into the mechanisms GPCR signaling, we must understand the vast degree of conformational dynamics that exists both within and between these states with atomic-level detail to decipher the complex atomic mechanisms underlying these processes. In this dissertation, we combine molecular dynamics with structural, biochemical and biophysical studies to bridge this gap. In Chapter 1, we identify TM6 as a steric selectivity filter that facilities discrimination between Gs (stimulatory) and Gi (inhibitory) protein coupling by GPCRs. In Chapter 2, we identify a unifying sequence motif found within the C-terminal tail of most GPCRs, and many non-GPCR membrane proteins, that contributes to high affinity arrestin coupling. In Chapter 3, we uncover the chemical and molecular basis of fentanyl-mediated arrestin biased signaling for the μ-opioid receptor. The sum of these studies provides atomistic insights into the processes of ligand binding, receptor activation, and effector coupling