Community recommendations on cryoEM data archiving and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

Community recommendations on cryoEM data archiving and validation: Outcomes of a wwPDB/EMDB workshop on cryoEM data management, deposition and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and consensus recommendations resulting from the workshop. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

Structure determination by cryoEM at 100 keV.

Electron cryomicroscopy can, in principle, determine the structures of most biological molecules but is currently limited by access, specimen preparation difficulties, and cost. We describe a purpose-built instrument operating at 100 keV-including advances in electron optics, detection, and processing-that makes structure determination fast and simple at a fraction of current costs. The instrument attains its theoretical performance limits, allowing atomic resolution imaging of gold test specimens and biological molecular structure determination in hours. We demonstrate its capabilities by determining the structures of eleven different specimens, ranging in size from 140 kDa to 2 MDa, using a fraction of the data normally required. CryoEM with a microscope designed specifically for high-efficiency, on-the-spot imaging of biological molecules will expand structural biology to a wide range of previously intractable problems.

`Cryo-EM': electron cryomicroscopy, cryo electron microscopy or something else?

Structural biology continues to benefit from an expanding toolkit, which is helping to gain unprecedented insight into the assembly and organization of multi-protein machineries, enzyme mechanisms and ligand/inhibitor binding. During the last ten years, cryoEM has become widely available and has provided a major boost to structure determination of membrane proteins and large multi-protein complexes. Many of the structures have now been made available at resolutions around 2 Å, where fundamental questions regarding enzyme mechanisms can be addressed. Over the years, the abbreviation cryoEM has been understood to stand for different things. We wish the wider community to engage and clarify the definition of cryoEM so that the expanding literature involving cryoEM is unified.

Grocery Purchase Changes Were Associated With A North Carolina COVID-19 Food Assistance Incentive Program.

Many low-income Americans experience food insecurity, which may have been exacerbated by economic instability during the early stages of the COVID-19 pandemic in 2020. In this study we assessed the impact of Healthy Helping, a short-term fruit and vegetable incentive program aimed at alleviating food insecurity and improving diet quality for Supplemental Nutrition Assistance Program participants, on grocery purchases, using transaction data from a large supermarket chain in North Carolina. We compared Healthy Helping participants' purchases of key food groups before and during the program with purchases by control shoppers participating in federal food assistance programs during the same period. Healthy Helping enrollment was associated with a $26.95 increase in monthly spending on fruit, vegetables, nuts, and legumes-an increase of 2.5 grams of fiber per 1,000 kilocalories purchased-and other shifts in the composition of food purchases, relative to control shoppers. These findings suggest that the program increased healthy food purchases while also increasing dollar sales at participating retailers. On average, participants did not use the full benefit; future research should explore factors associated with non- or underuse of benefits, to inform program design and outreach.

Activity-dependent plasticity and spinal cord stimulation for motor recovery following spinal cord injury.

Spinal cord injuries lead to permanent physical impairment despite most often being anatomically incomplete disruptions of the spinal cord. Remaining connections between the brain and spinal cord create the potential for inducing neural plasticity to improve sensorimotor function, even many years after injury. This narrative review provides an overview of the current evidence for spontaneous motor recovery, activity-dependent plasticity, and interventions for restoring motor control to residual brain and spinal cord networks via spinal cord stimulation. In addition to open-loop spinal cord stimulation to promote long-term neuroplasticity, we also review a more targeted approach: closed-loop stimulation. Lastly, we review mechanisms of spinal cord neuromodulation to promote sensorimotor recovery, with the goal of advancing the field of rehabilitation for physical impairments following spinal cord injury.

On the reduction in the effects of radiation damage to two-dimensional crystals of organic and biological molecules at liquid-helium temperature.

We have studied the fading of electron diffraction spots from two-dimensional (2D) crystals of paraffin (C44H90), purple membrane (bacteriorhodopsin) and aquaporin 4 (AQP4) at stage temperatures between 4K and 100K. We observed that the diffraction spots at resolutions between 3 Å and 20 Å fade more slowly at liquid-helium temperatures compared to liquid-nitrogen temperatures, by a factor of between 1.2 and 1.8, depending on the specimens. If the reduction in the effective rate of radiation damage for 2D crystals at liquid-helium temperature (as measured by spot fading) can be shown to extend to macromolecular assemblies embedded in amorphous ice, this would suggest that valuable improvements to electron cryomicroscopy (cryoEM) of biological specimens could be made by reducing the temperature of the specimens under irradiation below what is obtainable using standard liquid-nitrogen cryostats.

Interaction of human erythrocyte catalase with air-water interface in cryoEM.

One of the key goals in single-particle cryo-microscopy is to obtain a uniform distribution of particle orientations, so that the three-dimensional structure has isotropic resolution in Fourier space. A common problem arises from the interaction of protein molecules with the air-water interface that exists on both surfaces of the thin film of liquid that is formed prior to plunge-freezing into liquid ethane. Some proteins and other macromolecular complexes are disrupted by interaction with the air-water interface. Other proteins or macromolecules either become concentrated through their interaction with the interface or are excluded because they bind strongly to some other part of the grid or the filter paper used in blotting. In this paper, the interaction of human erythrocyte catalase with the air-water interface is investigated and minimized by the addition of certain detergents. Detergents can form an amphipathic monolayer at the air-water interface that creates a barrier and leaves the molecules free to adopt a variety of orientations, thus facilitating the 3D structure determination. These results suggest that further characterization and development of detergents for cryo-microscopy plunge-freezing would be useful.

Glassy Carbon Neural Interface for Chronic Epidural Stimulation in Rats with Cervical Spinal Cord Injury.

There is growing evidence on the efficacy of electrical stimulation delivered via spinal neural interfaces to improve functional recovery following spinal cord injury. For such interfaces, carbon-based neural arrays are fast becoming recognized as a compelling material and platform due to biocompatibility and long-term electrochemical stability. Here, we introduce the design, fabrication, and in vivo characterization of a novel cervical epidural implant with carbon-based surface electrodes. Through finite element analysis and mechanical load tests, we demonstrated that the array could safely withstand loads applied to it during implantation and natural movement of the subject with minimal stress levels. Furthermore, the long-term in vivo performance of this neural array consisting of glassy carbon surface electrodes was investigated through acute and chronic spinal motor evoked potential recordings and electrode impedance tests in rats. We demonstrated stable stimulation performance for at least four weeks in a rat model of spinal cord injury. Lastly, we found that impedance measurements on all carbon-based spinal arrays were generally stable over time up to four weeks after implantation, with a slight increase in impedance in subsequent weeks when tested in spinally injured rats. Taken together, this study demonstrated the potential for carbon-based electrodes as a spinal neural interface to accelerate both mechanistic research and functional restoration in animal models of spinal cord injury.

Association of a Fruit and Vegetable Subsidy Program With Food Purchases by Individuals With Low Income in the US.

Importance: Food insecurity is associated with a less healthy diet. The Supplemental Nutrition Assistance Program (SNAP) is associated with reduced food insecurity, but benefit levels may be insufficient for beneficiaries to afford healthy foods. Objective: To evaluate whether participation in SuperSNAP, a program that provides an additional $40 per month for the purchase of fruits and vegetables with no added sugar, sodium, or fat to SNAP beneficiaries, is associated with changes in food purchasing. Design, Setting, and Participants: This longitudinal cohort study used data from transaction records of a large supermarket chain with approximately 500 stores located across North Carolina from October 2019 to April 2020. Participants were SNAP beneficiaries. Exposure: SuperSNAP participation. Main Outcomes and Measures: Monthly spending on all fruits, vegetables, legumes, and nuts (primary outcome); spending on less healthy food categories; and spending on sugar-sweetened beverages as 1 category of less healthy foods. Monthly data on purchases by SNAP beneficiaries before and during SuperSNAP participation were compared with data from SNAP beneficiaries not enrolled in the program who shopped at the same stores. Overlap weighting (a propensity score-based method) was used to account for confounding, and linear mixed-effects models were fitted with random effects to account for repeated measures and clustering by store. Results: The study included 667 SuperSNAP participants and 33 246 SNAP beneficiaries who did not use SuperSNAP but shopped in the same stores; 436 SuperSNAP participants had preintervention data and were included in the main analysis. SuperSNAP participation was associated with increased monthly purchases of fruits, vegetables, nuts, and legumes ($31.84; 95% CI, $31.27-$32.42; P < .001; 294.52 oz; 95% CI, 288.84-300.20 oz; P < .001). Only a small increase in spending on less healthy food categories compared with the SNAP beneficiaries who did not use SuperSNAP ($1.60; 95% CI, $0.67-$2.53; P < .001) was observed. As total spending increased, the proportion of total food and beverage spending on less healthy foods significantly decreased (difference, 4.51%; 95% CI, 4.27%-4.74%; P < .001). Monthly spending on sugar-sweetened beverages decreased (difference, $1.83; 95% CI, $1.30-$2.36; P < .001). Conclusions and Relevance: In this cohort study, participation in SuperSNAP was associated with meaningful increases in healthy food purchasing. Subsequent studies should investigate whether healthy food incentive programs improve health outcomes.

Characterization of Patients in a Lifestyle Medicine Practice.

Objective. Lifestyle medicine is a relatively new specialty within medicine. The aim of this report is to characterize patients who present to a lifestyle medicine clinical practice. Methods. LifeStyle Medical Centers is an independent, community-based practice; this report is based on over 3200 patients within this practice. Most of the data presented were obtained from an intake questionnaire developed by the practice to provide background and screening particularly relevant to lifestyle medicine, including areas such as stress, sleep, physical activity, health knowledge, motivation, weight loss history and goals, and smoking. Results. Patients who present for lifestyle care come with varied goals, past histories, and current lifestyle issues. Many express a very high level of motivation to lose an unrealistically large amount of weight. The prevalence of physical inactivity, inadequate sleep, high stress, and risk of depression are high, yet the importance of these to health and well-being are often not recognized by the patient. Over 90% of the cost of care was covered by insurance. Conclusions. Having a better understanding of the patients who come to a lifestyle medicine clinic will help practices better design their lifestyle programs and guide lifestyle medicine providers to better engagement and care of their patients.

Defocus-dependent Thon-ring fading.

The brightness of modern Schottky field-emission guns can produce electron beams that have very high spatial coherence, especially for the weak-illumination conditions that are used for single-particle electron cryo-microscopy in structural biology. Even so, many users have observed defocus-dependent Thon-ring fading that has led them to restrict their data collection strategy to imaging with relatively small defocus values. In this paper, we reproduce the observation of defocus-dependent Thon-ring fading and produce a quantitative analysis and clear explanation of its causes. We demonstrate that a major cause is the delocalization of high-resolution Fourier components outside the field of view of the camera. We also show that, to correctly characterize the phenomenon, it is important to make a correction for linear magnification anisotropy. Even when the anisotropy is quite small, it is present at all defocus values before circular averaging of the Thon rings, as is also true before merging data from particles in many orientations. Under the conditions used in this paper, which are typical of those used in single-particle electron cryomicroscopy, fading of the Thon rings due to source coherence is negligible. The principal conclusion is that much higher values of defocus can be used to record images than is currently thought to be possible, keeping in mind that the above-mentioned delocalization of Fourier components will ultimately become a limitation. This increased understanding should give electron microscopists the confidence to use higher amounts of defocus to allow, for example, better visibility of their particles and Ewald sphere correction.

Carbon footprint impact of the choice of inhalers for asthma and COPD.

In the 1990s, metered dose inhalers (MDIs) containing chlorofluorocarbons were replaced with dry-powder inhalers (DPIs) and MDIs containing hydrofluorocarbons (HFCs). While HFCs are not ozone depleting, they are potent greenhouse gases. Annual carbon footprint (CO2e), per patient were 17 kg for Relvar-Ellipta/Ventolin-Accuhaler; and 439 kg for Seretide-Evohaler/Ventolin-Evohaler. In 2017, 70% of all inhalers sold in England were MDI, versus 13% in Sweden. Applying the Swedish DPI and MDI distribution to England would result in an annual reduction of 550 kt CO2e. The lower carbon footprint of DPIs should be considered alongside other factors when choosing inhalation devices.

The energy dependence of contrast and damage in electron cryomicroscopy of biological molecules.

We have measured the dependence on electron energy of elastic and inelastic scattering cross-sections from carbon, over the energy range that includes 100 keV to 300 keV. We also compared quantitatively the radiation damage to bacteriorhodopsin and paraffin (C44H90) at 100 keV and 300 keV by observing the fading of the diffraction spots from two-dimensional crystals as a function of electron fluence. The elastic cross-section is 2.01 - fold greater at 100 keV than at 300 keV, whereas the radiation damage increased by only 1.57. This implies that the amount of useful information from diffraction patterns or images of most biological structures should be 25% greater using 100 keV rather than 300 keV electrons. Using these measurements, we calculate the energy dependence of the available information per unit damage for a specimen of a particular thickness, which we call the "information coefficient." This allows us to determine the optimal energy for imaging a biological specimen of a given thickness. We find that for most single particle cryoEM specimens, 100 keV provides not only the highest potential for information per unit damage, but would also simplify the instrument while retaining the potential to reach high resolution with a minimum of data. These measurements will help guide the development and use of electron cryomicroscopes for biology.