Biophysical Modeling of Synaptic Plasticity.

Dendritic spines are small, bulbous compartments that function as postsynaptic sites and undergo intense biochemical and biophysical activity. The role of the myriad signaling pathways that are implicated in synaptic plasticity is well studied. A recent abundance of quantitative experimental data has made the events associated with synaptic plasticity amenable to quantitative biophysical modeling. Spines are also fascinating biophysical computational units because spine geometry, signal transduction, and mechanics work in a complex feedback loop to tune synaptic plasticity. In this sense, ideas from modeling cell motility can inspire us to develop multiscale approaches for predictive modeling of synaptic plasticity. In this article, we review the key steps in postsynaptic plasticity with a specific focus on the impact of spine geometry on signaling, cytoskeleton rearrangement, and membrane mechanics. We summarize the main experimental observations and highlight how theory and computation can aid our understanding of these complex processes.Expected final online publication date for the Annual Review of Biophysics, Volume 53 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Full-length direct RNA sequencing uncovers stress-granule dependent RNA decay upon cellular stress.

Cells react to stress by triggering response pathways, leading to extensive alterations in the transcriptome to restore cellular homeostasis. The role of RNA metabolism in shaping the cellular response to stress is vital, yet the global changes in RNA stability under these conditions remain unclear. In this work, we employ direct RNA sequencing with nanopores, enhanced by 5' end adaptor ligation, to comprehensively interrogate the human transcriptome at single-molecule and nucleotide resolution. By developing a statistical framework to identify robust RNA length variations in nanopore data, we find that cellular stress induces prevalent 5' end RNA decay that is coupled to translation and ribosome occupancy. Unlike typical RNA decay models in normal conditions, we show that stress-induced RNA decay is dependent on XRN1 but does not depend on removal of the poly(A) tail. We observed that RNAs undergoing decay are predominantly enriched in the stress granule transcriptome. Inhibition of stress granule formation via genetic ablation of G3BP1 and G3BP2 fully rescues RNA length and suppresses stress-induced decay. Our findings reveal RNA decay as a key determinant of RNA metabolism upon cellular stress and dependent on stress-granule formation.

Cristae formation is a mechanical buckling event controlled by the inner mitochondrial membrane lipidome.

Cristae are high-curvature structures in the inner mitochondrial membrane (IMM) that are crucial for ATP production. While cristae-shaping proteins have been defined, analogous lipid-based mechanisms have yet to be elucidated. Here, we combine experimental lipidome dissection with multi-scale modeling to investigate how lipid interactions dictate IMM morphology and ATP generation. When modulating phospholipid (PL) saturation in engineered yeast strains, we observed a surprisingly abrupt breakpoint in IMM topology driven by a continuous loss of ATP synthase organization at cristae ridges. We found that cardiolipin (CL) specifically buffers the inner mitochondrial membrane against curvature loss, an effect that is independent of ATP synthase dimerization. To explain this interaction, we developed a continuum model for cristae tubule formation that integrates both lipid and protein-mediated curvatures. This model highlighted a snapthrough instability, which drives IMM collapse upon small changes in membrane properties. We also showed that cardiolipin is essential in low-oxygen conditions that promote PL saturation. These results demonstrate that the mechanical function of cardiolipin is dependent on the surrounding lipid and protein components of the IMM.

ActuAtor, a Listeria-inspired molecular tool for physical manipulation of intracellular organizations through de novo actin polymerization.

Form and function are often interdependent throughout biology. Inside cells, mitochondria have particularly attracted attention since both their morphology and functionality are altered under pathophysiological conditions. However, directly assessing their causal relationship has been beyond reach due to the limitations of manipulating mitochondrial morphology in a physiologically relevant manner. By engineering a bacterial actin regulator, ActA, we developed tools termed "ActuAtor" that inducibly trigger actin polymerization at arbitrary subcellular locations. The ActuAtor-mediated actin polymerization drives striking deformation and/or movement of target organelles, including mitochondria, Golgi apparatus, and nucleus. Notably, ActuAtor operation also disperses non-membrane-bound entities such as stress granules. We then implemented ActuAtor in functional assays, uncovering the physically fragmented mitochondria being slightly more susceptible to degradation, while none of the organelle functions tested are morphology dependent. The modular and genetically encoded features of ActuAtor should enable its application in studies of the form-function interplay in various intracellular contexts.

Membrane mechanics dictate axonal morphology and function.

Axons are thought to be ultrathin membrane cables of a relatively uniform diameter, designed to conduct electrical signals, or action potentials. Here, we demonstrate that unmyelinated axons are not simple cylindrical tubes. Rather, axons have nanoscopic boutons repeatedly along their length interspersed with a thin cable with a diameter of ∼60 nm like pearls-on-a-string. These boutons are only ∼200 nm in diameter and do not have synaptic contacts or a cluster of synaptic vesicles, hence non-synaptic. Our in silico modeling suggests that axon pearling can be explained by the mechanical properties of the membrane including the bending modulus and tension. Consistent with modeling predictions, treatments that disrupt these parameters like hyper- or hypo-tonic solutions, cholesterol removal, and non-muscle myosin II inhibition all alter the degree of axon pearling, suggesting that axon morphology is indeed determined by the membrane mechanics. Intriguingly, neuronal activity modulates the cholesterol level of plasma membrane, leading to shrinkage of axon pearls. Consequently, the conduction velocity of action potentials becomes slower. These data reveal that biophysical forces dictate axon morphology and function and that modulation of membrane mechanics likely underlies plasticity of unmyelinated axons.

The ins and outs of membrane bending by intrinsically disordered proteins.

Membrane curvature is essential to diverse cellular functions. While classically attributed to structured domains, recent work illustrates that intrinsically disordered proteins are also potent drivers of membrane bending. Specifically, repulsive interactions among disordered domains drive convex bending, while attractive interactions drive concave bending, creating membrane-bound, liquid-like condensates. How might disordered domains that contain both repulsive and attractive domains affect curvature? Here, we examined chimeras that combined attractive and repulsive interactions. When the attractive domain was closer to the membrane, its condensation amplified steric pressure among repulsive domains, leading to convex curvature. In contrast, when the repulsive domain was closer to the membrane, attractive interactions dominated, resulting in concave curvature. Further, a transition from convex to concave curvature occurred with increasing ionic strength, which reduced repulsion while enhancing condensation. In agreement with a simple mechanical model, these results illustrate a set of design rules for membrane bending by disordered proteins.

Modelling the economic burden of SARS-CoV-2 infection in health care workers in four countries.

Health care workers (HCWs) experienced greater risk of SARS-CoV-2 infection during the COVID-19 pandemic. This study applies a cost-of-illness (COI) approach to model the economic burden associated with SARS-CoV-2 infections among HCWs in five low- and middle-income sites (Kenya, Eswatini, Colombia, KwaZulu-Natal province, and Western Cape province of South Africa) during the first year of the pandemic. We find that not only did HCWs have a higher incidence of COVID-19 than the general population, but in all sites except Colombia, viral transmission from infected HCWs to close contacts resulted in substantial secondary SARS-CoV-2 infection and death. Disruption in health services as a result of HCW illness affected maternal and child deaths dramatically. Total economic losses attributable to SARS-CoV-2 infection among HCWs as a share of total health expenditure ranged from 1.51% in Colombia to 8.38% in Western Cape province, South Africa. This economic burden to society highlights the importance of adequate infection prevention and control measures to minimize the risk of SARS-CoV-2 infection in HCWs.

Implementation of the 7-1-7 target for detection, notification, and response to public health threats in five countries: a retrospective, observational study.

BACKGROUND: Suboptimal detection and response to recent outbreaks, including COVID-19 and mpox (formerly known as monkeypox), have shown that the world is insufficiently prepared for public health threats. Routine monitoring of detection and response performance of health emergency systems through timeliness metrics has been proposed to evaluate and improve outbreak preparedness and contain health threats early. We implemented 7-1-7 to measure the timeliness of detection (target of ≤7 days from emergence), notification (target of ≤1 day from detection), and completion of seven early response actions (target of ≤7 days from notification), and we identified bottlenecks to and enablers of system performance. METHODS: In this retrospective, observational study, we conducted reviews of public health events in Brazil, Ethiopia, Liberia, Nigeria, and Uganda with staff from ministries of health and national public health institutes. For selected public health events occurring from Jan 1, 2018, to Dec 31, 2022, we calculated timeliness intervals for detection, notification, and early response actions, and synthesised identified bottlenecks and enablers. We mapped bottlenecks and enablers to Joint External Evaluation (second edition) indicators. FINDINGS: Of 41 public health events assessed, 22 (54%) met a target of 7 days to detect (median 6 days [range 0-157]), 29 (71%) met a target of 1 day to notify (0 days [0-24]), and 20 (49%) met a target of 7 days to complete all early response actions (8 days [0-72]). 11 (27%) events met the complete 7-1-7 target, with variation among event types. 25 (61%) of 41 bottlenecks to and 27 (51%) of 53 enablers of detection were at the health facility level, with delays to notification (14 [44%] of 32 bottlenecks) and response (22 [39%] of 56 bottlenecks) most often at an intermediate public health (ie, municipal, district, county, state, or province) level. Rapid resource mobilisation for responses (six [9%] of 65 enablers) from the national level enabled faster responses. INTERPRETATION: The 7-1-7 target is feasible to measure and to achieve, and assessment with this framework can identify areas for performance improvement and help prioritise national planning. Increased investments must be made at the health facility and intermediate public health levels for improved systems to detect, notify, and rapidly respond to emerging public health threats. FUNDING: Bill & Melinda Gates Foundation.

The road to achieving epidemic-ready primary health care.

Millions of avoidable deaths arising from the COVID-19 pandemic emphasise the need for epidemic-ready primary health care aligned with public health to identify and stop outbreaks, maintain essential services during disruptions, strengthen population resilience, and ensure health worker and patient safety. The improvement in health security from epidemic-ready primary health care is a strong argument for increased political support and can expand primary health-care capacities to improve detection, vaccination, treatment, and coordination with public health-needs that became more apparent during the pandemic. Progress towards epidemic-ready primary health care is likely to be stepwise and incremental, advancing when opportunity arises based on explicit agreement on a core set of services, improved use of external and national funds, and payment based in large part on empanelment and capitation to improve outcomes and accountability, supplemented with funding for core staffing and infrastructure and well designed incentives for health improvement. Health-care worker and broader civil society advocacy, political consensus, and bolstering government legitimacy could promote strong primary health care. Epidemic-ready primary health-care infrastructure that is able to help prevent and withstand the next pandemic will require substantial financial and structural reforms and sustained political and financial commitment. Governments, advocates, and bilateral and multilateral agencies should seize this window of opportunity before it closes.

Cristae formation is a mechanical buckling event controlled by the inner membrane lipidome.

Cristae are high curvature structures in the inner mitochondrial membrane (IMM) that are crucial for ATP production. While cristae-shaping proteins have been defined, analogous mechanisms for lipids have yet to be elucidated. Here we combine experimental lipidome dissection with multi-scale modeling to investigate how lipid interactions dictate IMM morphology and ATP generation. When modulating phospholipid (PL) saturation in engineered yeast strains, we observed a surprisingly abrupt breakpoint in IMM topology driven by a continuous loss of ATP synthase organization at cristae ridges. We found that cardiolipin (CL) specifically buffers the IMM against curvature loss, an effect that is independent of ATP synthase dimerization. To explain this interaction, we developed a continuum model for cristae tubule formation that integrates both lipid and protein-mediated curvatures. The model highlighted a snapthrough instability, which drives IMM collapse upon small changes in membrane properties. We also showed that CL is essential in low oxygen conditions that promote PL saturation. These results demonstrate that the mechanical function of CL is dependent on the surrounding lipid and protein components of the IMM.

Building National Health Security Through a Rapid Self-Assessment and Annual Operational Plan in Uganda, May to September 2021.

Uganda established a National Action Plan for Health Security in 2019, following a Joint External Evaluation (JEE) of International Health Regulations (2005) capacities in 2017. The action plan enhanced national health security awareness, but implementation efforts were affected by limited funding, excess of activities, and challenges related to monitoring and evaluation. To improve implementation, Uganda conducted a multisectoral health security self-assessment in 2021 using the second edition of the JEE tool and developed a 1-year operational plan. From 2017 to 2021, Uganda's composite ReadyScore improved by 20%, with improvement in 13 of the 19 technical areas. Indicator scores showing limited capacity declined from 30% to 20%, and indicators with no capacity declined from 10% to 2%. More indicators had developed (47% vs 40%), demonstrated (29% vs 20%), and sustained (2% vs 0%) capacities in 2021 compared with 2017. Using the self-assessment JEE scores, 72 specific activities from the International Health Regulations (2005) benchmarks tool were selected for inclusion in a 1-year operational plan (2021-2022). In contrast to the 264 broad activities in the 5-year national action plan, the operational plan prioritized a small number of activities to enable sectors to focus limited resources on implementation. While certain capacities improved before and during implementation of the action plan, countries may benefit from using short-term operational planning to develop realistic and actionable health security plans to improve health security capacities.

Spatiotemporal modelling reveals geometric dependence of AMPAR dynamics on dendritic spine morphology.

The modification of neural circuits depends on the strengthening and weakening of synaptic connections. Synaptic strength is often correlated to the density of the ionotropic, glutamatergic receptors, AMPARs, (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors) at the postsynaptic density (PSD). While AMPAR density is known to change based on complex biological signalling cascades, the effect of geometric factors such as dendritic spine shape, size and curvature remain poorly understood. In this work, we developed a deterministic, spatiotemporal model to study the dynamics of AMPARs during long-term potentiation (LTP). This model includes a minimal set of biochemical events that represent the upstream signalling events, trafficking of AMPARs to and from the PSD, lateral diffusion in the plane of the spine membrane, and the presence of an extrasynaptic AMPAR pool. Using idealized and realistic spine geometries, we show that the dynamics and increase of bound AMPARs at the PSD depends on a combination of endo- and exocytosis, membrane diffusion, the availability of free AMPARs and intracellular signalling interactions. We also found non-monotonic relationships between spine volume and the change in AMPARs at the PSD, suggesting that spines restrict changes in AMPARs to optimize resources and prevent runaway potentiation. KEY POINTS: Synaptic plasticity involves dynamic biochemical and physical remodelling of small protrusions called dendritic spines along the dendrites of neurons. Proper synaptic functionality within these spines requires changes in receptor number at the synapse, which has implications for downstream neural functions, such as learning and memory formation. In addition to being signalling subcompartments, spines also have unique morphological features that can play a role in regulating receptor dynamics on the synaptic surface. We have developed a spatiotemporal model that couples biochemical signalling and receptor trafficking modalities in idealized and realistic spine geometries to investigate the role of biochemical and biophysical factors in synaptic plasticity. Using this model, we highlight the importance of spine size and shape in regulating bound AMPA receptor dynamics that govern synaptic plasticity, and predict how spine shape might act to reset synaptic plasticity as a built-in resource optimization and regulation tool.

Mem3DG: Modeling membrane mechanochemical dynamics in 3D using discrete differential geometry.

Biomembranes adopt varying morphologies that are vital to cellular functions. Many studies use computational modeling to understand how various mechanochemical factors contribute to membrane shape transformations. Compared with approximation-based methods (e.g., finite element method [FEM]), the class of discrete mesh models offers greater flexibility to simulate complex physics and shapes in three dimensions; its formulation produces an efficient algorithm while maintaining coordinate-free geometric descriptions. However, ambiguities in geometric definitions in the discrete context have led to a lack of consensus on which discrete mesh model is theoretically and numerically optimal; a bijective relationship between the terms contributing to both the energy and forces from the discrete and smooth geometric theories remains to be established. We address this and present an extensible framework, Mem3DG, for modeling 3D mechanochemical dynamics of membranes based on discrete differential geometry (DDG) on triangulated meshes. The formalism of DDG resolves the inconsistency and provides a unifying perspective on how to relate the smooth and discrete energy and forces. To demonstrate, Mem3DG is used to model a sequence of examples with increasing mechanochemical complexity: recovering classical shape transformations such as 1) biconcave disk, dumbbell, and unduloid; and 2) spherical bud on spherical, flat-patch membrane; investigating how the coupling of membrane mechanics with protein mobility jointly affects phase and shape transformation. As high-resolution 3D imaging of membrane ultrastructure becomes more readily available, we envision Mem3DG to be applied as an end-to-end tool to simulate realistic cell geometry under user-specified mechanochemical conditions.

Role of the Triplet State and Protein Dynamics in the Formation and Stability of the Tryptophan Radical in an Apoazurin Mutant.

The protein, azurin, has enabled the study of the tryptophan radical. Upon UV excitation of tyrosine-deficient apoazurin and in the presence of a Co(III) electron acceptor, the neutral radical (W48•) is formed. The lifetime of W48• in apoazurin is 41 s, which is shorter than the lifetime of several hours in Zn-substituted azurin. Molecular dynamics simulations revealed enhanced fluctuations of apoazurin which likely destabilize W48•. The photophysics of W48 was investigated to probe the precursor state for ET. The phosphorescence intensity was eliminated in the presence of an electron acceptor while the fluorescence was unchanged; this quenching of the phosphorescence is attributed to ET. The kinetics associated with W48• were examined with a model that incorporates intersystem crossing, ET, deprotonation, and decay of the cation radical. The estimated rate constants for ET (6 × 106 s-1) and deprotonation (3 × 105 s-1) are in agreement with a photoinduced mechanism where W48• is derived from the triplet state. The triplet as the precursor state for ET was supported by photolysis of apoazurin with 280 nm in the absence and presence of triplet-absorbing 405 nm light. Absorption bands from the neutral radical were observed only in the presence of blue light.

Dendritic spine morphology regulates calcium-dependent synaptic weight change.

Dendritic spines act as biochemical computational units and must adapt their responses according to their activation history. Calcium influx acts as the first signaling step during postsynaptic activation and is a determinant of synaptic weight change. Dendritic spines also come in a variety of sizes and shapes. To probe the relationship between calcium dynamics and spine morphology, we used a stochastic reaction-diffusion model of calcium dynamics in idealized and realistic geometries. We show that despite the stochastic nature of the various calcium channels, receptors, and pumps, spine size and shape can modulate calcium dynamics and subsequently synaptic weight updates in a deterministic manner. Through a series of exhaustive simulations and analyses, we found that the calcium dynamics and synaptic weight change depend on the volume-to-surface area of the spine. The relationships between calcium dynamics and spine morphology identified in idealized geometries also hold in realistic geometries, suggesting that there are geometrically determined deterministic relationships that may modulate synaptic weight change.

Impact of a Newly Established Revolving Outbreak Investigation Fund on Timeliness of Response to Public Health Emergencies in Nigeria.

Timely access to emergency funding has been identified as a bottleneck for outbreak response in Nigeria. In February 2019, a new revolving outbreak investigation fund (ROIF) was established by the Nigeria Centre for Disease Control (NCDC). We abstracted the date of NCDC notification, date of verification, and date of response for 25 events that occurred prior to establishing the fund (April 2017 to August 2019) and for 8 events that occurred after establishing the fund (February to October 2019). The median time to notification (1 day) and to verification (0 days) did not change after establishing the ROIF, but the median time to response significantly decreased, from 6 days to 2 days (P = .003). Response to disease outbreaks was accelerated by access to emergency funding with a clear approval process. We recommend that the ROIF should be financed by the national government through budget allocation. Finally, development partners can provide financial support for the existing fund and technical assistance for protocol development toward financial accountability and sustainability.

Development of a simple and effective online training for health workers: results from a pilot in Nigeria.

BACKGROUND: Health workers (HWs) in Africa face challenges accessing and learning from existing online training opportunities. To address these challenges, we developed a modular, self-paced, mobile-ready and work-relevant online course covering foundational infection prevention and control (IPC) concepts. Here, we evaluate the first pilot of this course, conducted with HWs in Nigeria. METHODS: We used a learner-centered design and prototyping process to create a new approach to delivering online training for HWs. The resulting course comprised 10 self-paced modules optimized for use on mobile devices. Modules presented IPC vignettes in which learning was driven by short assessment questions with feedback. Learners were recruited by distributing a link to the training through Nigeria-based email lists, WhatsApp groups and similar networks of HWs, managers and allied professionals. The course was open to learners for 8 weeks. We tracked question responses and time on task with platform analytics and assessed learning gains with pre- and post-testing. Significance was evaluated with the Wilcoxon signed-rank test, and effect size was calculated using Cohen's d. RESULTS: Three hundred seventy-two learners, with roles across the health system, enrolled in the training; 59% completed all 10 modules and earned a certificate. Baseline knowledge of foundational IPC concepts was low, as measured by pre-test scores (29%). Post-test scores were significantly higher at 54% (effect size 1.22, 95% confidence interval 1.00-1.44). Learning gains were significant both among learners with low pre-test scores and among those who scored higher on the pre-test. We used the Net Promoter Score (NPS), a common user experience metric, to evaluate the training. The NPS was + 62, which is slightly higher than published scores of other self-paced online learning experiences. CONCLUSIONS: High completion rates, significant learning gains and positive feedback indicate that self-paced, mobile-ready training that emphasizes short, low-stakes assessment questions can be an effective, scalable way to train HWs who choose to enroll. Low pre-test scores suggest that there are gaps in IPC knowledge among this learner population.

Benchmarking ensemble docking methods in D3R Grand Challenge 4.

The discovery of new drugs is a time consuming and expensive process. Methods such as virtual screening, which can filter out ineffective compounds from drug libraries prior to expensive experimental study, have become popular research topics. As the computational drug discovery community has grown, in order to benchmark the various advances in methodology, organizations such as the Drug Design Data Resource have begun hosting blinded grand challenges seeking to identify the best methods for ligand pose-prediction, ligand affinity ranking, and free energy calculations. Such open challenges offer a unique opportunity for researchers to partner with junior students (e.g., high school and undergraduate) to validate basic yet fundamental hypotheses considered to be uninteresting to domain experts. Here, we, a group of high school-aged students and their mentors, present the results of our participation in Grand Challenge 4 where we predicted ligand affinity rankings for the Cathepsin S protease, an important protein target for autoimmune diseases. To investigate the effect of incorporating receptor dynamics on ligand affinity rankings, we employed the Relaxed Complex Scheme, a molecular docking method paired with molecular dynamics-generated receptor conformations. We found that Cathepsin S is a difficult target for molecular docking and we explore some advanced methods such as distance-restrained docking to try to improve the correlation with experiments. This project has exemplified the capabilities of high school students when supported with a rigorous curriculum, and demonstrates the value of community-driven competitions for beginners in computational drug discovery.

Reviewing Health Security Capacities in Nigeria Using the Updated WHO Joint External Evaluation and WHO Benchmarks Tool: Experience from a Country-Led Self-Assessment Exercise.

Across the world, the level of pandemic preparedness varies and no country is fully prepared to respond to all public health events. The International Health Regulations 2005 require state parties to develop core capacities to prevent, detect, and respond to public health events of international concern. In addition to annual self-assessment, these capacities are peer reviewed once every 5 years through the voluntary Joint External Evaluation (JEE). In this article, we share Nigeria's experience of conducting a country-led midterm self-assessment using a slightly modified application of the second edition of the World Health Organization (WHO) JEE and the new WHO benchmarks tool. Despite more stringent scoring criteria in the revised JEE tool, average scoring showed modest capacity improvements in 2019 compared with 2017. Of the 19 technical areas assessed, 11 improved, 5 did not change, and 3 had lower scores. No technical area attained the highest-level scoring of 5. Understanding the level of, and gaps in, pandemic preparedness enables state parties to develop plans to improve health security; the outcome of the assessment included the development of a 12-month operational plan. Countries need to intentionally invest in preparedness by using existing frameworks (eg, JEE) to better understand the status of their preparedness. This will ensure ownership of developed plans with shared responsibilities by all key stakeholders across all levels of government.