High-confidence 3D template matching for cryo-electron tomography.

Visual proteomics attempts to build atlases of the molecular content of cells but the automated annotation of cryo electron tomograms remains challenging. Template matching (TM) and methods based on machine learning detect structural signatures of macromolecules. However, their applicability remains limited in terms of both the abundance and size of the molecular targets. Here we show that the performance of TM is greatly improved by using template-specific search parameter optimization and by including higher-resolution information. We establish a TM pipeline with systematically tuned parameters for the automated, objective and comprehensive identification of structures with confidence 10 to 100-fold above the noise level. We demonstrate high-fidelity and high-confidence localizations of nuclear pore complexes, vaults, ribosomes, proteasomes, fatty acid synthases, lipid membranes and microtubules, and individual subunits inside crowded eukaryotic cells. We provide software tools for the generic implementation of our method that is broadly applicable towards realizing visual proteomics.

Structure and assembly of a bacterial gasdermin pore.

In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis1-3. Studies of human and mouse GSDM pores have revealed the functions and architectures of assemblies comprising 24 to 33 protomers4-9, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing more than 50 protomers. We determine a cryo-electron microscopy structure of a Vitiosangium bGSDM in an active 'slinky'-like oligomeric conformation and analyse bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning ß-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.

Cell-free biosynthesis combined with deep learning accelerates de novo-development of antimicrobial peptides.

Bioactive peptides are key molecules in health and medicine. Deep learning holds a big promise for the discovery and design of bioactive peptides. Yet, suitable experimental approaches are required to validate candidates in high throughput and at low cost. Here, we established a cell-free protein synthesis (CFPS) pipeline for the rapid and inexpensive production of antimicrobial peptides (AMPs) directly from DNA templates. To validate our platform, we used deep learning to design thousands of AMPs de novo. Using computational methods, we prioritized 500 candidates that we produced and screened with our CFPS pipeline. We identified 30 functional AMPs, which we characterized further through molecular dynamics simulations, antimicrobial activity and toxicity. Notably, six de novo-AMPs feature broad-spectrum activity against multidrug-resistant pathogens and do not develop bacterial resistance. Our work demonstrates the potential of CFPS for high throughput and low-cost production and testing of bioactive peptides within less than 24 h.

Structure and assembly of a bacterial gasdermin pore.

In response to pathogen infection, gasdermin (GSDM) proteins form membrane pores that induce a host cell death process called pyroptosis1-33. Studies of human and mouse GSDM pores reveal the functions and architectures of 24-33 protomers assemblies4-9, but the mechanism and evolutionary origin of membrane targeting and GSDM pore formation remain unknown. Here we determine a structure of a bacterial GSDM (bGSDM) pore and define a conserved mechanism of pore assembly. Engineering a panel of bGSDMs for site-specific proteolytic activation, we demonstrate that diverse bGSDMs form distinct pore sizes that range from smaller mammalian-like assemblies to exceptionally large pores containing >50 protomers. We determine a 3.3 Å cryo-EM structure of a Vitiosangium bGSDM in an active slinky-like oligomeric conformation and analyze bGSDM pores in a native lipid environment to create an atomic-level model of a full 52-mer bGSDM pore. Combining our structural analysis with molecular dynamics simulations and cellular assays, our results support a stepwise model of GSDM pore assembly and suggest that a covalently bound palmitoyl can leave a hydrophobic sheath and insert into the membrane before formation of the membrane-spanning ß-strand regions. These results reveal the diversity of GSDM pores found in nature and explain the function of an ancient post-translational modification in enabling programmed host cell death.

Sublytic gasdermin-D pores captured in atomistic molecular simulations.

Gasdermin-D (GSDMD) is the ultimate effector of pyroptosis, a form of programmed cell death associated with pathogen invasion and inflammation. After proteolytic cleavage by caspases, the GSDMD N-terminal domain (GSDMDNT) assembles on the inner leaflet of the plasma membrane and induces the formation of membrane pores. We use atomistic molecular dynamics simulations to study GSDMDNT monomers, oligomers, and rings in an asymmetric plasma membrane mimetic. We identify distinct interaction motifs of GSDMDNT with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylserine (PS) headgroups and describe their conformational dependence. Oligomers are stabilized by shared lipid binding sites between neighboring monomers acting akin to double-sided tape. We show that already small GSDMDNT oligomers support stable, water-filled, and ion-conducting membrane pores bounded by curled beta-sheets. In large-scale simulations, we resolve the process of pore formation from GSDMDNT arcs and lipid efflux from partial rings. We find that high-order GSDMDNT oligomers can crack under the line tension of 86 pN created by an open membrane edge to form the slit pores or closed GSDMDNT rings seen in atomic force microscopy experiments. Our simulations provide a detailed view of key steps in GSDMDNT-induced plasma membrane pore formation, including sublytic pores that explain nonselective ion flux during early pyroptosis.

Binding of SARS-CoV-2 Fusion Peptide to Host Endosome and Plasma Membrane.

During infection the SARS-CoV-2 virus fuses its viral envelope with cellular membranes of its human host. The viral spike (S) protein mediates both the initial contact with the host cell and the subsequent membrane fusion. Proteolytic cleavage of S at the S2' site exposes its fusion peptide (FP) as the new N-terminus. By binding to the host membrane, the FP anchors the virus to the host cell. The reorganization of S2 between virus and host then pulls the two membranes together. Here we use molecular dynamics (MD) simulations to study the two core functions of the SARS-CoV-2 FP: to attach quickly to cellular membranes and to form an anchor strong enough to withstand the mechanical force during membrane fusion. In eight 10 µs long MD simulations of FP in proximity to endosomal and plasma membranes, we find that FP binds spontaneously to the membranes and that binding proceeds predominantly by insertion of two short amphipathic helices into the membrane interface. Connected via a flexible linker, the two helices can bind the membrane independently, yet binding of one promotes the binding of the other by tethering it close to the target membrane. By simulating mechanical pulling forces acting on the C-terminus of the FP, we then show that the bound FP can bear forces up to 250 pN before detaching from the membrane. This detachment force is more than 10-fold higher than an estimate of the force required to pull host and viral membranes together for fusion. We identify a fully conserved disulfide bridge in the FP as a major factor for the high mechanical stability of the FP membrane anchor. We conclude, first, that the sequential binding of two short amphipathic helices allows the SARS-CoV-2 FP to insert quickly into the target membrane, before the virion is swept away after shedding the S1 domain connecting it to the host cell receptor. Second, we conclude that the double attachment and the conserved disulfide bridge establish the strong anchoring required for subsequent membrane fusion. Multiple distinct membrane-anchoring elements ensure high avidity and high mechanical strength of FP-membrane binding.

Introduction of sacubitril/valsartan in primary care follow-up of heart failure: a prospective observational study (THESEUS).

AIMS: Switch from angiotensin converting enzyme inhibitor treatment to sacubitril/valsartan (sac/val) is associated with benefit in heart failure with reduced ejection fraction (HFrEF). Reports on management of this switch are largely based on randomized controlled trials, retrospective analyses, and hospital-based care, while patients with chronic heart failure (CHF) are frequently followed-up in primary care. The THESEUS study aimed to characterize the transition to sac/val and early maintenance period of HFrEF in primary care. METHOD AND RESULTS: THESEUS was a prospective, observational, non-interventional study, performed at primary care sites throughout Switzerland. Patient characteristics, sac/val transition, and maintenance were reported at study enrolment and approximately 3 and 6 months after sac/val initiation. The primary endpoint was achievement of 200 mg BID sac/val with maintenance for ≥12 weeks. Secondary outcomes included dosing regimens, healthcare utilization in the 6 months prior to sac/val initiation and during the study, patient well-being, safety, and tolerability. Fifty-eight patients with CHF were enrolled from 45 primary care centres. Six patients were excluded, and 19 achieved the primary endpoint (36.5%, Achievers). Non-Achievers underwent fewer titration steps than Achievers (1.9 ± 0.9 vs. 3.1 ± 1.4). In both groups, patient well-being improved and the percentage of New York Heart Association III patients decreased. Healthcare utilization decreased (19% vs. 30.8% in the 6 months pre-enrolment period). The most frequent reasons for target dose non-achievement were asymptomatic and symptomatic hypotension (15.3% and 12.1%, respectively). CONCLUSIONS: Results from THESEUS suggest that transition to sac/val is manageable in primary care, with a safety profile corresponding to reports from specialized heart failure care.

QCD Coupling from a Nonperturbative Determination of the Three-Flavor Λ Parameter.

We present a lattice determination of the Λ parameter in three-flavor QCD and the strong coupling at the Z pole mass. Computing the nonperturbative running of the coupling in the range from 0.2 to 70 GeV, and using experimental input values for the masses and decay constants of the pion and the kaon, we obtain Λ_{MS[over ¯]}^{(3)}=341(12) MeV. The nonperturbative running up to very high energies guarantees that systematic effects associated with perturbation theory are well under control. Using the four-loop prediction for Λ_{MS[over ¯]}^{(5)}/Λ_{MS[over ¯]}^{(3)} yields α_{MS[over ¯]}^{(5)}(m_{Z})=0.11852(84).

Testing trivializing maps in the Hybrid Monte Carlo algorithm.

We test a recent proposal to use approximate trivializing maps in a field theory to speed up Hybrid Monte Carlo simulations. Simulating the CPN-1 model, we find a small improvement with the leading order transformation, which is however compensated by the additional computational overhead. The scaling of the algorithm towards the continuum is not changed. In particular, the effect of the topological modes on the autocorrelation times is studied.

Differentiation of arginine vasopressin antagonistic effects by selective V2 versus dual V2/V1a receptor blockade in a preclinical heart failure model.

Arginine vasopressin (AVP) is increased in patients with heart failure (HF). Its actions are linked to free water reabsorption (V2-) and arteriolar vasoconstriction (V1a receptor). AVP can exacerbate the cardiorenal syndrome with excess fluid retention and afterload increase. Tolvaptan (TOL; selective V2 antagonist) and Conivaptan (CON; dual V1a/V2 antagonist) are two AVP antagonists that counteract the action of AVP with distinct profiles. We investigated the therapeutic effects of CON and TOL in an acute HF model. Mongrel dogs were paced continuously at 220 beats/min. After 14 days, the animals underwent acute testing. Dogs were instrumented to measure cardiac output, blood pressure, pulmonary artery pressure, and left ventricular dP/dtmax. Additionally, during the acute experiments, vasopressin was infused intravenously (4 mU/kg/min) to achieve constant and controlled pathophysiological levels of AVP. Subsequently, animals received either CON or TOL (n = 6; 0.1-mg/kg bolus). There were no significant differences in effect on mean arterial pressure, dP/dtmax, central venous pressure, and urine output between CON and TOL. In contrast, cardiac output increased by 0.15 l/min after CON and decreased by 0.6 l/min after TOL (P < 0.01). Accordingly, the total peripheral resistance increased after TOL by 250 dyn*s/cm and decreased after CON by 125 dyn*s/cm (P < 0.01). In conclusion, it was demonstrated that in an acute HF model, CON lowered, whereas TOL increased afterload. The results suggest that dual V1a/V2 blockade in the acute HF setting could be beneficial compared with selective V2 blockade. Chronic experiments are needed to determine whether this finding can translate into a sustained clinical advantage.

Speeding up parallel tempering simulations.

We discuss methods that allow us to increase the step size in a parallel tempering simulation of statistical models and test them at the example of the three-dimensional Heisenberg spin glass. We find an overall speedup of about 2 for contemporary lattices.

Effects of combined inhibition of the Na+-H+ exchanger and angiotensin-converting enzyme in rats with congestive heart failure after myocardial infarction.

1 We investigated the single vs the combined long-term inhibition of Na(+)-H(+) exchanger-1 (NHE-1) and ACE in rats with congestive heart failure induced by myocardial infarction (MI). 2 Rats with MI were randomized to receive either placebo, cariporide (3000 p.p.m. via chow), ramipril (1 mg kg(-1) day(-1) via drinking water) or their combination for 18 weeks starting on day 3 after surgery. 3 Cardiac morphology and function was assessed by echocardiography and by means of a 2.0 F conductance catheter to determine left ventricular (LV) pressure volume relationships. 4 MI for 18 weeks resulted in an increase in LV end-diastolic diameter (LVDed) in the placebo-treated group when compared to sham (placebo: 1.1+/-0.04 cm; sham: 0.86+/-0.01; P<0.05). Combined inhibition of NHE-1 and ACE, but not the monotherapies, significantly reduced LVDed (1.02+/-0.02 cm). 5 Preload recruitable stroke work (PRSW), dp/dt(max) (parameter of systolic function) and end-diastolic pressure volume relationship (EDPVR, diastolic function) were significantly impaired in placebo-treated MI group (PRSW: 39+/-7 mmHg; dp/dt(max): 5185+/-363 mmHg s(-1); EDPVR: 0.042+/-0.001 mmHg microl(-1); all P<0.05). Cariporide treatment significantly improved PRSW (64+/-7 mmHg), dp/dt(max) (8077+/-525 mmHg s(-1)) and EDPVR (0.026+/-0.014 mmHg microl(-1)), and reduced cardiac hypertrophy in rats with MI. Combined inhibition of NHE-1 and ACE had even a more pronounced effect on PRSW (72+/-5 mmHg) and EDPVR (0.026+/-0.014 mmHg microl(-1)), as well as cardiac hypertrophy that, however, did not reach statistical significance compared to cariporide treatment alone. 6 The NHE-1 inhibitor cariporide significantly improved LV remodeling and function in rats with congestive heart failure induced by MI. The effect of cariporide was comparable or tended to be stronger (e.g. systolic function) compared to ramipril. Combined treatment with cariporide and ramipril tended to be more effective on LV remodeling in rats with heart failure than the single treatments. Thus, inhibition of the NHE-1 may be a promising novel therapeutic approach for the treatment of congestive heart failure.

School-based health center viability: application of the COPC model.

Providing effective primary and preventive health care to adolescents is challenging. Traditional medical and nursing models have had little success in guiding health care for the individual adolescent. A community-based model would aim programs and intervention at high-risk adolescents identified as needing treatment. Community-Oriented Primary Care (COPC) bridges the gap between primary care and public health to evaluate and improve the delivery of health care to identified populations. The COPC model was used to develop and implement an adolescent school-based health center in a southwestern border community. The four process steps based on the COPC model include: identifying the community of interest, identifying the health problem, developing and implementing intervention(s), and conducting ongoing evaluation. Key stakeholders from the medical, educational, and consumer groups were invited to participate in the first and second steps. Representatives of various school adolescent subcultures formed a group of promotores (health promoters) to monitor, advise, and contribute to the process. The Dartmouth COOP survey was selected to identify health concerns and risks of the targeted high school. The promotores prepared and administered the survey to 1,116 students. Results were used to design the initial health center program components using an interdisciplinary team to implement interventions aimed at the major health concerns and risks identified by the COOP. A short feedback loop contributed to program refinements, and ongoing evaluation continues to shape the practice of health care providers in the school-based health care center.