Single-particle EM reveals the higher-order domain architecture of soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is the primary nitric oxide (NO) receptor in mammals and a central component of the NO-signaling pathway. The NO-signaling pathways mediate diverse physiological processes, including vasodilation, neurotransmission, and myocardial functions. sGC is a heterodimer assembled from two homologous subunits, each comprised of four domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length sGC. We used single-particle electron microscopy to obtain the structure of the complete sGC heterodimer and determine its higher-order domain architecture. Overall, the protein is formed of two rigid modules: the catalytic dimer and the clustered Per/Art/Sim and heme-NO/O₂-binding domains, connected by a parallel coiled coil at two hinge points. The quaternary assembly demonstrates a very high degree of flexibility. We captured hundreds of individual conformational snapshots of free sGC, NO-bound sGC, and guanosine-5′-[(α,β)-methylene]triphosphate-bound sGC. The molecular architecture and pronounced flexibility observed provides a significant step forward in understanding the mechanism of NO signaling.Nitric oxide (NO) has emerged as an integral signaling molecule in biology. Soluble guanylate cyclase (sGC), the primary receptor of NO in mammals, binds NO via an Fe^II heme cofactor leading to a several hundred-fold increase in 3,5-cyclic guanosine monophosphate (cGMP) synthesis. cGMP then acts as a second messenger, targeting phosphodiesterases, ion-gated channels, and cGMP-dependent protein kinases. These target proteins go on to regulate many critical physiological functions including vasodilation, platelet aggregation, neurotransmission, and myocardial functions (1, 2). Disruptions in NO signaling have been linked to hypertension, erectile dysfunction, neurodegeneration, stroke, and heart disease (3, 4). sGC has been the focus of small-molecule modulators of activity for therapeutic advantage. Riociguat, which is a stimulator of sGC, has recently been approved for treatment of pulmonary hypertension (5). However, the mechanistic details underlying the modulation of sGC catalytic activity by NO and other small molecules remain largely unknown. Determining the structure of the full-length sGC, free and in complex with NO, is therefore a prerequisite to understanding its function and for the design and improvement of therapeutics for treatment of diseases involving the NO/cGMP pathway.The most extensively studied and physiologically relevant isoform of sGC is the 150-kDa heterodimer containing one α1 and one β1 subunit. Each subunit is comprised of four modular domains: the N-terminal heme-NO/O₂–binding (H-NOX), the Per/Arnt/Sim (PAS), the helical, and the C-terminal catalytic domain (Fig. 1). No structure of the complete holoenzyme is available to date, and its absence precludes answering key questions such as how NO occupancy of the N-terminal β H-NOX sensor domain is communicated to the C-terminal cyclase domain. Atomic models of isolated sGC domains have been obtained by X-ray crystallography or homology modeling (6–9) (Fig. 1). The arrangement of and interactions between these domains have been further studied using a variety of techniques, including mutational and truncation studies, Förster resonance energy transfer (FRET), resonance Raman spectroscopy, chemical cross-linking, small-angle X-ray scattering (SAXS), and hydrogen deuterium exchange (HDX) (10–14). These studies have been used to propose a variety of models for the mechanisms of action of sGC, but the lack of a comprehensive 3D structure of the sGC holoenzyme has so far impeded a confident assignment of domain hierarchy.Open in a separate windowFig. 1.sGC domain organization and X-ray crystallographic models. Each subunit contains four modular domains; α1 domains are shown in shades of gray, and β1 domains are shown in color. The H-NOX domain of the β1 subunit contains the heme cofactor, shown in red. Structures for Rattus norvegicus are modeled based on previously solved crystal structures of homologous domains (Materials and Methods). The H-NOX structures are modeled from a standalone Nostoc sp. PCC 7120 H-NOX domain (PDB: 2O09) (6). The PAS and helical domains are modeled on individual domain truncations. The PAS domain is based on the PAS domain from Manduca sexta (PDB: 4GJ4) (7), and the helical domain is based on the β1 R. norvegicus structure (3HLS) (8). The catalytic domain is the Homo sapiens α1β1 crystal structure (PDB: 3UVJ) (9).Here, we used EM to determine the first structure of the heterodimeric sGC holoenzyme. Fitting of the domain crystal structures into the EM reconstruction provides a detailed model for the higher-order architecture and quaternary organization of sGC and is consistent with all reported biochemical data. We obtained hundreds of individual 3D reconstructions of full-length Rattus norvegicus sGC using automated high-throughput single-particle electron microscopy (15, 16). The structures correspond to various snapshots of the enzyme and describe the conformational trajectory of this highly flexible protein. sGC is assembled from two ridged units: the smaller unit comprises the dimeric catalytic domain, and the larger unit is built from the clustering of the PAS and H-NOX domains. The helical domains form a dimeric parallel coiled coil that flexibly connects the two modules. These modules swing freely in relation to each other thereby allowing the structure to access a wide range of conformations. Strikingly, some of these conformations allow the N-terminal H-NOX domain to contact the C-terminal catalytic domain indicating the possibility of a direct allosteric control mechanism. We also obtained reconstructions of sGC in complex with NO as well as with guanosine-5′-[(α,β)-methylene]triphosphate (GPCPP), a noncyclizable analog of the natural substrate GTP, both in the presence and absence of NO. The overall domain architecture and range of the accessible conformations of these complexes are similar to the unbound sGC state, suggesting that ligand binding induces small-scale intradomain conformational changes mediated by flexibility transitions at two key linker points.     

Prevalence of blood donation eligibility in Australia: A population survey

Background

Reliable estimates of the population proportion eligible to donate blood are needed by blood collection agencies to model the likely impact of changes in eligibility criteria and inform targeted population-level education, recruitment, and retention strategies. In Australia, the sole estimate was calculated 10+ years ago. With several subsequent changes to the eligibility criteria, an updated estimate is required.

Study Design and Methods

We conducted a cross-sectional national population survey to estimate eligibility for blood donation. Respondents were aged 18+ and resident in Australia. Results were weighted to obtain a representative sample of the population.

Results

Estimated population prevalence of blood donation eligibility for those aged 18–74 was 57.3% (95% CI 55.3–59.3). The remaining 42.7% (95% CI 40.7–44.7) were either temporarily (25.3%, 95% CI 23.5–27.2) or permanently ineligible (17.4%, 95% CI 16.1–18.9). Of those eligible at the time of the survey, that is, with the UK geographic deferral for variant Creutzfeldt-Jakob disease included, (52.9%, 95% CI 50.8–54.9), 14.2% (95% CI 12.3–16.3) reported donating blood within the previous 2 years. Eligibility was higher among men (62.6%, 95% CI 59.6–65.6) than women (52.8%, 95% CI 50.1–55.6). The most common exclusion factor was iron deficiency/anemia within the last 6 months; 3.8% (95% CI 3.2–4.6) of the sample were ineligible due to this factor alone.

Discussion

We estimate that approximately 10.5 million people (57.3% of 18–74-year-olds) are eligible to donate blood in Australia. Only 14.2% of those eligible at the time of survey reported donating blood within the previous 2 years, indicating a large untapped pool of potentially eligible blood donors.     

Physicians’ Attitudes and Experiences with Medical Aid in Dying in Colorado: a “Hidden Population” Survey

BackgroundApproximately 20% of the US population live in states where MAiD is a legal, though highly contentious, practice. Little generalizable data exists on the experiences of MAiD providers who comprise a small, and intentionally hidden, population.ObjectiveTo examine the nature, extent, and consequences of physicians’ participation in MAiD.DesignAn anonymous, multi-wave, mailed survey (RR= 55%).ParticipantsAn enriched sample (n=583) of Colorado physicians caring for potential MAiD patients.Main MeasuresPhysician willingness, preparedness, and participation in a continuum of MAiD activities. Other outcomes include the effects of providing MAiD and the barriers physicians face related to MAiD.Key ResultsOverall, 81.1% of respondents were willing to discuss MAiD with a patient, 88.3% to refer for MAiD, 46.3% to be a consultant, and 28.1% to be an attending. Fewer felt prepared to discuss MAiD (54.4%), provide a MAiD referral (62.8%), be a consultant (30.7%), or be an attending (18.0%). More than half of respondents (52.3%) had discussed MAiD with a patient, 27.3% provided a MAiD referral, 12.8% had been a MAiD consultant, and 8.5% had been a MAiD attending. Among MAiD consultants and attendings, 75% reported that their most recent MAiD case was emotionally fulfilling and professionally rewarding, though 75% also reported that it was time consuming and 46.9% reported that it was ethically challenging. Common barriers to physician participation in MAiD include lack of knowledge about MAiD (46.8%), the emotional (45.6%) and time (41.7%) investments, and ethical concerns (41.7%).ConclusionsMany physicians in our sample are both willing and prepared to discuss MAiD with patients and to provide MAiD referrals. Fewer are prepared and willing to serve as an attending or consultant and fewer have provided these services. MAID consultants and attendings largely report the experience to be emotionally fulfilling and professionally rewarding, but all respondents reported multiple barriers to participation.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11606-021-07300-8.KEY WORDS: physician assisted death, physician assisted suicide, medical aid in the dying     

Bulimia associated with repeated spontaneous abortion

We report a case demonstrating a previously undescribed association between bulimia and repeated spontaneous abortion. It is also the first report of bulimia in a British Sikh woman. Her history reveals features of multiimpulsivity and demonstrates an unusually early onset of bulimia associated with an organic condition, asthma.     

Protective effects of pharmacological therapies in animal models of multiple sclerosis: a review of studies 2014–2019

Multiple sclerosis(MS)is an inflammatory demyelinating disease of the central nervous system.The disability caused by inflammatory demyelination clinically dominates the early stages of relapsing-remitting MS and is reversible.Once there is considerable loss of axons,MS patients enter a secondary progressive stage.Disease-modifying drugs currently in use for MS suppress the immune system and reduce relapse rates but are not effective in the progressive stage.Various animal models of MS(mostly mouse and rat)have been established and proved useful in studying the disease process and response to therapy.The experimental autoimmune encephalomyelitis animal studies reviewed here showed that a chronic progressive disease can be induced by immunization with appropriate amounts of myelin oligodendrocyte glycoprotein together with mycobacterium tuberculosis and pertussis toxin in Freund's adjuvant.The clinical manifestations of autoimmune encephalomyelitis disease were prevented or reduced by treatment with certain pharmacological agents given prior to,at,or after peak disease,and the agents had protective effects as shown by inhibiting demyelination and damage to neurons,axons and oligodendrocytes.In the cuprizone-induced toxicity animal studies,the pharmacological agents tested were able to promote remyelination and increase the number of oligodendrocytes when administered therapeutically or prophylactically.A monoclonal IgM antibody protected axons in the spinal cord and preserved motor function in animals inoculated with Theiler's murine encephalomyelitis virus.In all these studies the pharmacological agents were administered singly.A combination therapy may be more effective,especially using agents that target neuroinflammation and neurodegeneration,as they may exert synergistic actions.     

The natural history of disordered eating behavior and attitudes in adult women

This paper reports a survey of natural history of eating behavior and attitudes to weight and shape in British women attending a family planning clinic. Sixty-two women completed a battery of standardized tests, followed by a second questionnaire 18 months later. This rescreening represents the first report of an adult sample not self-defined as ill. The Eating Attitudes Test (EAT)-26 total score showed stability over time (r =.68) but a/so showed a nonsignificant increase in the number of “cases.” The self-reported weight and desired weight increased slightly but statistically over the whole sample in the follow-up period. Analysis of group variances showed evidence of a heterogeneity of variance which was more marked than evidence of group mean effects between responders and nonresponders. The self-reported heights were stable but self-report of historical weight limits showed impossible inconsistencies such that any data related to these must be interpreted with caution. We recommend that future follow-up studies report analysis of loss to follow-up both by group means and variances, that inconsistent responses be counted and explored, and that both group mean changes over time and correlations be reported since they convey different information.     

Disentangling Family Life and Hair Pulling: Trichotillomania and Relatedness

ABSTRACT

Trichotillomania (hair pulling) remains a relatively unknown form of body-focused repetitive behavior (BFRB). Sufferers tend to conceal both the action and its effects from others because of stigmatization, which is strong in both public and domestic spheres. Negative responses from close family members can add significantly to the suffering. Based on fieldwork in the United Kingdom and United States, we explore how hair pulling troubles ties even among close family members. We show why ethnographic methods reveal impacts of hair pulling that structured assessments do not yet capture and argue for a more nuanced study of BFRBs through anthropologies of relatedness.