Atoms to Phenotypes: Molecular Design Principles of Cellular Energy Metabolism.

We report a 100-million atom-scale model of an entire cell organelle, a photosynthetic chromatophore vesicle from a purple bacterium, that reveals the cascade of energy conversion steps culminating in the generation of ATP from sunlight. Molecular dynamics simulations of this vesicle elucidate how the integral membrane complexes influence local curvature to tune photoexcitation of pigments. Brownian dynamics of small molecules within the chromatophore probe the mechanisms of directional charge transport under various pH and salinity conditions. Reproducing phenotypic properties from atomistic details, a kinetic model evinces that low-light adaptations of the bacterium emerge as a spontaneous outcome of optimizing the balance between the chromatophore's structural integrity and robust energy conversion. Parallels are drawn with the more universal mitochondrial bioenergetic machinery, from whence molecular-scale insights into the mechanism of cellular aging are inferred. Together, our integrative method and spectroscopic experiments pave the way to first-principles modeling of whole living cells.

Expanding the Optogenetics Toolkit by Topological Inversion of Rhodopsins.

Targeted manipulation of activity in specific populations of neurons is important for investigating the neural circuit basis of behavior. Optogenetic approaches using light-sensitive microbial rhodopsins have permitted manipulations to reach a level of temporal precision that is enabling functional circuit dissection. As demand for more precise perturbations to serve specific experimental goals increases, a palette of opsins with diverse selectivity, kinetics, and spectral properties will be needed. Here, we introduce a novel approach of "topological engineering"-inversion of opsins in the plasma membrane-and demonstrate that it can produce variants with unique functional properties of interest for circuit neuroscience. In one striking example, inversion of a Channelrhodopsin variant converted it from a potent activator into a fast-acting inhibitor that operates as a cation pump. Our findings argue that membrane topology provides a useful orthogonal dimension of protein engineering that immediately permits as much as a doubling of the available toolkit.

A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.

Comparison of adaptation characteristics between visually- and memory-guided saccades.

Saccade adaptation plays a crucial role in maintaining saccade accuracy. The behavioral characteristics and neural mechanisms of saccade adaptation for an externally cued movement, such as visually-guided saccades (VGS), are well studied in non-human primates. In contrast, little is known about the saccade adaptation of an internally driven movement, such as memory-guided saccades (MGS), which are guided by visuospatial working memory. As the oculomotor plant changes due to growth, aging, or skeletomuscular problems, both types of saccades need to be adapted. Do both saccade types engage a common adaptation mechanism? In this study, we compared the characteristics of amplitude decrease adaptation in MGS with VGS in non-human primates. We found that the adaptation speed was faster for MGS than for VGS. Saccade duration changed during MGS adaptation, while saccade peak velocity changed during VGS adaptation. We also compared the adaptation field, that is, the gain change for saccade amplitudes other than the adapted. The gain change for MGS declines on both smaller and larger sides of adapted amplitude, more rapidly for larger than smaller amplitudes, while the decline in VGS was reversed. Thus, the differences between VGS and MGS adaptation characteristics support the previously suggested hypothesis that the adaptation mechanisms of VGS and MGS are distinct. Furthermore, the result suggests that the MGS adaptation site is a brain structure that influences saccade duration, while the VGS adaptation site influences saccade peak velocity. These results should be beneficial for future neurophysiological experiments.

AMBERff at Scale: Multimillion-Atom Simulations with AMBER Force Fields in NAMD.

All-atom molecular dynamics (MD) simulations are an essential structural biology technique with increasing application to multimillion-atom systems, including viruses and cellular machinery. Classical MD simulations rely on parameter sets, such as the AMBER family of force fields (AMBERff), to accurately describe molecular motion. Here, we present an implementation of AMBERff for use in NAMD that overcomes previous limitations to enable high-performance, massively parallel simulations encompassing up to two billion atoms. Single-point potential energy comparisons and case studies on model systems demonstrate that the implementation produces results that are as accurate as running AMBERff in its native engine.

Advanced Formulation Approaches for Emerging Therapeutic Technologies.

In addition to proteins, discussed in the Chapter "Advances in Vaccine Adjuvants: Nanomaterials and Small Molecules", there are a wide range of alternatives to small molecule active ingredients. Cells, extracellular vesicles, and nucleic acids in particular have attracted increasing research attention in recent years. There are now a number of products on the market based on these emerging technologies, the most famous of which are the mRNA-based vaccines against SARS-COV-2. These advanced therapeutic moieties are challenging to formulate however, and there remain significant challenges for their more widespread use. In this chapter, we consider the potential and bottlenecks for developing further medical products based on these systems. Cells, extracellular vesicles, and nucleic acids will be discussed in terms of their mechanism of action, the key requirements for translation, and how advanced formulation approaches can aid their future development. These points will be presented with selected examples from the literature, and with a focus on the formulations which have made the transition to clinical trials and clinical products.

Effect of a community-based primary healthcare programme on adverse pregnancy outcomes in Northern Ghana.

BACKGROUND: Pregnancy complications and adverse birth outcomes are among the major contributors to poor maternal and child health. Mothers in remote communities are at higher risk of adverse birth outcomes due to constraints in access to healthcare services. In Ghana, a community-based primary healthcare programme called the Ghana Essential Health Interventions Programme (GEHIP) was implemented in a rural region to help strengthen primary healthcare delivery and improve maternal and child healthcare services delivery. This study assessed the effect of this programme on adverse pregnancy outcomes. METHODS: Baseline and end-line survey data from reproductive-aged women from the GEHIP project were used in this analysis. Difference-in-differences and logistic regressions were used to examine the impact and equity effect of GEHIP on adverse pregnancy outcomes using household wealth index and maternal educational attainment as equity measures. The analysis involves the comparison of project baseline and end-line outcomes in intervention and non-intervention districts. RESULTS: The intervention had a significant effect in the reduction of adverse pregnancy outcomes (OR = 0.96, 95% CI:0.93-0.99). Although disadvantaged groups experience larger reductions in adverse pregnancy outcomes, controlling for covariates, there was no statistically significant equity effect of GEHIP on adverse pregnancy outcomes using either the household wealth index (OR = 0.99, 95% CI:0.85-1.16) or maternal educational attainment (OR = 0.68, 95% CI: 0.44-1.07) as equity measures. CONCLUSION: GEHIP's community-based healthcare programme reduced adverse pregnancy outcomes but no effect on relative equity was established. Factoring in approaches for targeting disadvantaged populations in the implementation of community-based health programs is crucial to ensuring equity in health outcomes.

Equity in maternal healthcare utilisation in Ghana: Do community-based primary health care programmes matter?

OBJECTIVE: Improving equity in the use of maternal health services in rural and remote communities is critical to meeting the Sustainable Development Goals targets on maternal and child health. This study examines the effect of a community-based primary healthcare strengthening programme on improving the utilisation of antenatal care (ANC4+), skilled delivery and health facility delivery. METHODS: Baseline and endline survey data of women of reproductive age for intervention and comparison districts were used to examine the equity impact of the Ghana Essential Health Interventions Programme (GEHIP) on antenatal care visits, skilled delivery and health facility delivery. The Wagstaff extension of the concentration index and regression models are used to assess equity effects of the programme on the utilisation of these services by wealth index and educational attainment. RESULTS: Coverage rates increased for both intervention and comparison districts, but were generally higher in intervention districts than comparison districts at endline (90% vs. 88% for ANC4+, 88% vs. 84% for skilled delivery and 93% vs. 88% for health facility delivery). Only ANC4+ showed a statistically significant positive treatment effect of the intervention (Dif-in-Dif = 0.071, p-value = 0.010). Equity analysis showed a mixed picture with intervention districts achieving significant equity improvement for skilled delivery for both wealth index and maternal education but only education equity for health facility delivery, while comparison districts achieved both wealth and education equity improvements for two indicators (health facility delivery and skilled delivery). No significant equity/inequity effects were found for ANC4+. CONCLUSION: Efforts to improve community-based healthcare access have been associated with improved coverage of maternal health services; however, the effect on improving equity in service coverage is mixed. Results indicate a need to extend community-based primary health care development beyond general improvements in access to ensure equity in the coverage of maternal and child health services that such programmes provide.

An Interim Analysis of the First 102 Patients Treated in the Prospective Vertebral Augmentation Sacroplasty Fracture Registry.

PURPOSE: To evaluate the efficacy of sacroplasty for treating sacral insufficiency fractures, including the effect on pain relief, patient function and adverse event rates in an as-treated on-label prospective data registry. MATERIALS AND METHODS: Observational data including patient reported outcomes (PROs), patient characteristics, osteoporosis treatment, fracture duration, cause of sacral fractures and image guidance used for treatment were collected for patients undergoing sacroplasty. The PROs were collected at baseline then at one, three, and at six months following the procedure. The primary outcomes were pain as measured by the Numerical Rating Scale (NRS) and function as measured by the Roland Morris Disability Questionnaire (RMDQ). Secondary outcomes included adverse events, cement leakage, new neurologic events, readmissions and death. RESULTS: The interim results for the first 102 patients included significant pain reduction with mean pain improvement scores at six months decreasing from 7.8 to 0.9 (P < .001) and significant improvement in function with mean RMDQ scores improving from 17.7 to 5.2 (P < .001). Most procedures were performed under fluoroscopy (58%). There was cement leakage in 17.7% of the subjects but only one adverse event which was a new neurologic deficit related to cement extravasation. The readmission rate was 16% mostly due to additional back pain and fractures and there were no subject deaths. CONCLUSIONS: Sacroplasty with cement augmentation for acute, subacute and chronic painful sacral insufficiency fractures caused by osteoporosis or neoplastic disorders results in highly significant improvements in pain and function with very low rate of procedural related adverse events.

Serum neurofilament light chain measurements following nerve trauma.

BACKGROUND: Optimal functional recovery following peripheral nerve injuries (PNIs) is dependent upon early recognition and prompt referral to specialist centres for appropriate surgical intervention. Technologies which facilitate the early detection of PNI would allow faster referral rates and encourage improvements in patient outcomes. Serum Neurofilament light chain (NfL) measurements are cheaper to perform, easier to access and interpret than many conventional methods used for nerve injury diagnosis, such as electromyography and/or magnetic resonance imaging assessments, but changes in serum NfL levels following traumatic PNI have not been investigated. This pre-clinical study aimed to determine whether serum NfL levels can: (1) detect the presence of a nerve trauma and (2) delineate between different severities of nerve trauma. METHODS: A rat sciatic nerve crush and common peroneal nerve crush were implemented as controlled animal models of nerve injury. At 1-, 3-, 7- and 21-days post-injury, serum samples were retrieved for analysis using the SIMOA® NfL analyser kit. Nerve samples were also retrieved for histological analysis. Static sciatic index (SSI) was measured at regular time intervals following injury. RESULTS: Significant 45-fold and 20-fold increases in NfL serum levels were seen 1-day post-injury following sciatic and common peroneal nerve injury, respectively. This corresponded with an eightfold higher volume of axons injured in the sciatic compared to the common peroneal nerve (p < .001). SSI measurements post-injury revealed greater reduction in function in the sciatic crush group compared with the common peroneal crush group. CONCLUSIONS: NfL serum measurements represent a promising method for detecting traumatic PNI and stratifying their severity. Clinical translation of these findings could provide a powerful tool to improve the surgical management of nerve-injured patients.

Structural basis of co-translational quality control by ArfA and RF2 bound to ribosome.

Quality control mechanisms intervene appropriately when defective translation events occur, in order to preserve the integrity of protein synthesis. Rescue of ribosomes translating on messenger RNAs that lack stop codons is one of the co-translational quality control pathways. In many bacteria, ArfA recognizes stalled ribosomes and recruits the release factor RF2, which catalyses the termination of protein synthesis. Although an induced-fit mechanism of nonstop mRNA surveillance mediated by ArfA and RF2 has been reported, the molecular interaction between ArfA and RF2 in the ribosome that is responsible for the mechanism is unknown. Here we report an electron cryo-microscopy structure of ArfA and RF2 in complex with the 70S ribosome bound to a nonstop mRNA. The structure, which is consistent with our kinetic and biochemical data, reveals the molecular interactions that enable ArfA to specifically recruit RF2, not RF1, into the ribosome and to enable RF2 to release the truncated protein product in this co-translational quality control pathway. The positively charged C-terminal domain of ArfA anchors in the mRNA entry channel of the ribosome. Furthermore, binding of ArfA and RF2 induces conformational changes in the ribosomal decoding centre that are similar to those seen in other protein-involved decoding processes. Specific interactions between residues in the N-terminal domain of ArfA and RF2 help RF2 to adopt a catalytically competent conformation for peptide release. Our findings provide a framework for understanding recognition of the translational state of the ribosome by new proteins, and expand our knowledge of the decoding potential of the ribosome.

Impact of community health interventions on maternal and child health indicators in the upper east region of Ghana.

BACKGROUND: This paper reports on results of a health system strengthening implementation research initiative conducted the Upper East Region of northern Ghana. Transformative interventions to accelerate and strengthen the health delivery were implemented that included empowering community leaders and members to actively participate in health delivery, strengthening the referral systems through the provision of community transport systems, providing basic medical equipment to community clinics, and improving the skills of critical health staff through training. METHODS: A mixed method design was used to evaluate the impact of the interventions. A quantitative evaluation employed a flexible research design to test the effects of various component activities of the project. To assess impact, a pre-versus-post randomized cluster survey design was used. Qualitative research was conducted with focus group data and individual in depth interviews to gauge the views of various stakeholders associated with the implementation process. RESULTS: After intervention, significant improvements in key maternal and child health indicators such as antenatal and postnatal care coverage were observed and increases in the proportion of deliveries occurring in health facilities and assisted by skilled health personnel relative to pre-intervention conditions. There was also increased uptake of oral rehydration salts (ORS) for treatment of childhood diarrhoea, as well as marked reductions in the incidence of upper respiratory infections (URI). CONCLUSIONS: A pre-and post-evaluation of impact suggests that the programme had a strong positive impact on the functioning of primary health care. Findings are consistent with the proposition that the coverage and content of the Ghana Community-based Health Planning and Services programme was improved by program interventions and induced discernable changes in key indicators of health system performance.

The Impact of Childhood Mortality on Fertility in Rural Tanzania: Evidence From the Ifakara and Rufiji Health and Demographic Surveillance Systems.

This manuscript examines the relationship between child mortality and subsequent fertility using longitudinal data on births and childhood deaths occurring among 15,291 Tanzanian mothers between 2000 and 2015. Generalized hazard regression analyses assess the effect of child loss on the hazard of conception, adjusting for child-level, mother-level, and contextual covariates. Results show that time to conception is most reduced if an index child dies during the subsequent birth interval, representing the combined effect of biological and volitional replacement. Deaths occurring during prior birth intervals were associated with accelerated time to conception during future intervals, consistent with hypothesized insurance effects of anticipating future child loss, but this effect is smaller than replacement effects. The analysis reveals that residence in areas of relatively high child mortality is associated with hastened parity progression, again consistent with the insurance hypothesis. Investigation of high-order interactions suggests that insurance effects tend to be greater in low-mortality communities, replacement effects tend to be stronger in high-mortality community contexts, and wealthier families tend to exhibit a weaker insurance response but a stronger replacement response to childhood mortality relative to poorer families.

Undiagnosing St Joan: She Does Not Need a Medical or Psychiatric Diagnosis.

ABSTRACT: This article traces the history of Joan of Arc through her brief life that includes leading an army in defense of France at the age of 17 and ending with her death at the stake at the age of 19. In her activities, St Joan reported that she was guided by voices and visions in which she communicated with venerated spiritual figures such as St Michael and St Margaret. Questions have arisen about the nature of these experiences, and various medical and psychiatric diagnoses have been offered by contemporary experts. In our effort to evaluate the diagnostic proposals, we have examined the incidence of voices and visions in the Middle Ages, and we have followed that with a review of nonpathologic voice-hearing in our own era. We then move on to an analysis of some proposed medical and psychiatric diagnoses, all of which we find unconvincing. With this background, we argue that St Joan does not warrant a medical or psychiatric diagnosis. Such a conclusion, however, leaves us with another issue, that of Joan's achievements. How do we understand an adolescent being able to lead an army? Addressing this question proves more difficult than deciding whether St Joan warrants a diagnosis. In addition to her achievements in the war against Britain, Joan of Arc stands out as both the most documented person in Western civilization up until her era, and as the only person who has been both condemned and canonized by the Catholic Church.

Local Administration of Minocycline Improves Nerve Regeneration in Two Rat Nerve Injury Models.

Peripheral nerve injuries are quite common and often require a surgical intervention. However, even after surgery, patients do not often regain satisfactory sensory and motor functions. This, in turn, results in a heavy socioeconomic burden. To some extent, neurons can regenerate from the proximal nerve stump and try to reconnect to the distal stump. However, this regenerating capacity is limited, and depending on the type and size of peripheral nerve injury, this process may not lead to a positive outcome. To date, no pharmacological approach has been used to improve nerve regeneration following repair surgery. We elected to investigate the effects of local delivery of minocycline on nerve regeneration. This molecule has been studied in the central nervous system and was shown to improve the outcome in many disease models. In this study, we first tested the effects of minocycline on SCL 4.1/F7 Schwann cells in vitro and on sciatic nerve explants. We specifically focused on the Schwann cell repair phenotype, as these cells play a central role in orchestrating nerve regeneration. Finally, we delivered minocycline locally in two different rat models of nerve injury, a sciatic nerve transection and a sciatic nerve autograft, demonstrating the capacity of local minocycline treatment to improve nerve regeneration.

Culture Conditions for Human Induced Pluripotent Stem Cell-Derived Schwann Cells: A Two-Centre Study.

Adult human Schwann cells represent a relevant tool for studying peripheral neuropathies and developing regenerative therapies to treat nerve damage. Primary adult human Schwann cells are, however, difficult to obtain and challenging to propagate in culture. One potential solution is to generate Schwann cells from human induced pluripotent stem cells (hiPSCs). Previously published protocols, however, in our hands did not deliver sufficient viable cell numbers of hiPSC-derived Schwann cells (hiPSC-SCs). We present here, two modified protocols from two collaborating laboratories that overcome these challenges. With this, we also identified the relevant parameters to be specifically considered in any proposed differentiation protocol. Furthermore, we are, to our knowledge, the first to directly compare hiPSC-SCs to primary adult human Schwann cells using immunocytochemistry and RT-qPCR. We conclude the type of coating to be important during the differentiation process from Schwann cell precursor cells or immature Schwann cells to definitive Schwann cells, as well as the amounts of glucose in the specific differentiation medium to be crucial for increasing its efficiency and the final yield of viable hiPSC-SCs. Our hiPSC-SCs further displayed high similarity to primary adult human Schwann cells.

An Enhanced Audiologic Protocol for Early Identification of Conductive Hearing Loss in Patients with Cleft Palate.

OBJECTIVE: To characterize the onset and prevalence of conductive hearing loss (CHL) in pediatric patients with cleft palate (CP) prior to palatoplasty with an enhanced audiologic protocol. DESIGN: Retrospective cohort study. SETTING: Multidisciplinary cleft and craniofacial clinic at a tertiary care center. PATIENTS: Patients with CP who received audiologic workup pre-operatively. Patients with bilateral permanent hearing loss, expiration prior to palatoplasty, or no pre-operative data were excluded. INTERVENTIONS: Patients with CP born February 2019 to November 2019 who passed newborn hearing screening (NBHS) received audiologic testing at 9 months of age (standard protocol). Patients born December 2019 to September 2020 underwent testing prior to 9 months of age (enhanced protocol). MAIN OUTCOME MEASURES: Age of identification of CHL in patients after implementation of the enhanced audiologic protocol. RESULTS: The number of patients who passed their NBHS in the standard protocol (n = 14, 54%) and the enhanced protocol (n = 25, 66%) did not differ. Infants who passed their NBHS, but demonstrated hearing loss on subsequent audiologic testing did not differ between enhanced (n = 25, 66%) and standard cohort (n = 14, 54%). Of patients who passed NBHS in the enhanced protocol, 48% (n = 12) had CHL identified by 3 months, and 20% (n = 5) by 6 months of age. With the enhanced protocol, patients who did not undergo additional testing post NBHS significantly dropped from 44.9% (n = 22) to 4.2% (n = 2) (P < .0001). CONCLUSION: Even with passed NBHS, CHL is still present for infants with CP pre-operatively. Earlier and more frequent testing for this population is recommended.

Decisional style, sleepiness, and online responsiveness.

As sleep problems can impair quality of work, an online questionnaire was used to examine relationships between sleepiness and decision making while obtaining unobtrusive indices of performance. Participants (N = 344) completed the Insomnia Severity Index, Epworth Sleepiness Scale, and the Melbourne Decision Making Questionnaire in a Qualtrics survey while reporting mobile phone use. Qualtrics recorded the time and the number of clicks required to complete each page of the survey. Multiple regression indicated that insomnia was associated with daytime sleepiness and Hypervigilance, and mobile phone use before bed. Participants with moderate sleepiness required a greater number of clicks to complete the questionnaire. Greater sleepiness was associated with longer times to complete these self-assessment tasks. Clinically significant sleepiness produces changes in performance that can be detected from online responsivity. As sleepy individuals can be appreciably and quantitatively slower in performing subjective self-assessment tasks, this argues for objective measures of sleepiness and automated interventions and the design of systems that allow better quality sleep.Practitioner summary: Work can require processing of electronic messages, but 24/7 accessibility increases workload, causes fatigue and potentially creates security risks. Although most studies use people's self-reports, this study monitors time and clicks required to complete self-assessment rating scales. Sleepiness affected online responsivity, decreasing online accuracy and increasing response times and hypervigilance.

#COVIDisAirborne: AI-enabled multiscale computational microscopy of delta SARS-CoV-2 in a respiratory aerosol.

We seek to completely revise current models of airborne transmission of respiratory viruses by providing never-before-seen atomic-level views of the SARS-CoV-2 virus within a respiratory aerosol. Our work dramatically extends the capabilities of multiscale computational microscopy to address the significant gaps that exist in current experimental methods, which are limited in their ability to interrogate aerosols at the atomic/molecular level and thus obscure our understanding of airborne transmission. We demonstrate how our integrated data-driven platform provides a new way of exploring the composition, structure, and dynamics of aerosols and aerosolized viruses, while driving simulation method development along several important axes. We present a series of initial scientific discoveries for the SARS-CoV-2 Delta variant, noting that the full scientific impact of this work has yet to be realized.

A combined experimental and computational framework to evaluate the behavior of therapeutic cells for peripheral nerve regeneration.

Recent studies have explored the potential of tissue-mimetic scaffolds in encouraging nerve regeneration. One of the major determinants of the regenerative success of cellular nerve repair constructs (NRCs) is the local microenvironment, particularly native low oxygen conditions which can affect implanted cell survival and functional performance. In vivo, cells reside in a range of environmental conditions due to the spatial gradients of nutrient concentrations that are established. Here we evaluate in vitro the differences in cellular behavior that such conditions induce, including key biological features such as oxygen metabolism, glucose consumption, cell death, and vascular endothelial growth factor secretion. Experimental measurements are used to devise and parameterize a mathematical model that describes the behavior of the cells. The proposed model effectively describes the interactions between cells and their microenvironment and could in the future be extended, allowing researchers to compare the behavior of different therapeutic cells. Such a combinatorial approach could be used to accelerate the clinical translation of NRCs by identifying which critical design features should be optimized when fabricating engineered nerve repair conduits.