Interpretability of high-resolution transmission electron microscopy images.

High-resolution electron microscopy is a well-suited tool for characterizing the nanoscale structure of materials. However, the interaction of the sample and the high-energy electrons of the beam can often have a detrimental impact on the sample structure. This effect can only be alleviated by decreasing the number of electrons to which the sample is exposed but will come at the cost of a decreased signal-to-noise ratio in the resulting image. Images with low signal to noise ratios are often challenging to interpret as parts of the sample with a low interaction with the electron beam are reproduced with very low contrast. Here we suggest simple measures as alternatives to the conventional signal-to-noise ratio and investigate how these can be used to predict the interpretability of the electron microscopy images. We test the models on a sample consisting of gold nanoparticles supported on a cerium dioxide substrate. The models are evaluated based on series of images acquired at varying electron dose.

Beam induced heating in electron microscopy modeled with machine learning interatomic potentials.

We develop a combined theoretical and experimental method for estimating the amount of heating that occurs in metallic nanoparticles that are being imaged in an electron microscope. We model the thermal transport between the nanoparticle and the supporting material using molecular dynamics and equivariant neural network potentials. The potentials are trained to Density Functional Theory (DFT) calculations, and we show that an ensemble of potentials can be used as an estimate of the errors the neural network make in predicting energies and forces. This can be used both to improve the networks during the training phase, and to validate the performance when simulating systems too big to be described by DFT. The energy deposited into the nanoparticle by the electron beam is estimated by measuring the mean free path of the electrons and the average energy loss, both are done with Electron Energy Loss Spectroscopy (EELS) within the microscope. In combination, this allows us to predict the heating incurred by a nanoparticle as a function of its size, its shape, the support material, and the electron beam energy and intensity.

Amyloid-ß-induced dendritic spine elimination requires Ca2+-permeable AMPA receptors, AKAP-Calcineurin-NFAT signaling, and the NFAT target gene Mdm2.

Alzheimer's Disease (AD) is associated with brain accumulation of synaptotoxic amyloid-ß (Aß) peptides produced by the proteolytic processing of amyloid precursor protein (APP). Cognitive impairments associated with AD correlate with dendritic spine and excitatory synapse loss, particularly within the hippocampus. In rodents, soluble Aß oligomers impair hippocampus-dependent learning and memory, promote dendritic spine loss, inhibit NMDA-type glutamate receptor (NMDAR)-dependent long-term potentiation (LTP), and promote synaptic depression (LTD), at least in part through activation of the Ca2+-CaM-dependent phosphatase calcineurin (CaN). Yet, questions remain regarding Aß-dependent postsynaptic CaN signaling specifically at the synapse to mediate its synaptotoxicity. Here, we use pharmacologic and genetic approaches to demonstrate a role for postsynaptic signaling via A kinase-anchoring protein 150 (AKAP150)-scaffolded CaN in mediating Aß-induced dendritic spine loss in hippocampal neurons from rats and mice of both sexes. In particular, we found that Ca2+-permeable AMPA-type glutamate receptors (CP-AMPARs), which were previously shown to signal through AKAP-anchored CaN to promote both LTD and Aß-dependent inhibition of LTP, are also required upstream of AKAP-CaN signaling to mediate spine loss via overexpression of APP containing multiple mutations linked to familial, early-onset AD and increased Aß production. In addition, we found that the CaN-dependent nuclear factor of activated T-cells (NFAT) transcription factors are required downstream to promote Aß-mediated dendritic spine loss. Finally, we identified the E3-ubiquitin ligase Mdm2, which was previously linked to LTD and developmental synapse elimination, as a downstream NFAT target gene upregulated by Aß whose enzymatic activity is required for Aß-mediated spine loss.Significance Statement Impaired hippocampal function and synapse loss are hallmarks of AD linked to Aß oligomers. Aß exposure acutely blocks hippocampal LTP and enhances LTD and chronically leads to dendritic spine synapse loss. In particular, Aß hijacks normal plasticity mechanisms, biasing them toward synapse weakening/elimination, with previous studies broadly linking CaN phosphatase signaling to this synaptic dysfunction. However, we do not understand how Aß engages signaling specifically at synapses. Here we elucidate a synapse-to-nucleus signaling pathway coordinated by the postsynaptic scaffold protein AKAP150 that is activated by Ca2+ influx through CP-AMPARs and transduced to nucleus by CaN-NFAT signaling to transcriptionally upregulate the E3-ubiquitin ligase Mdm2 that is required for Aß-mediated spine loss. These findings identify Mdm2 as potential therapeutic target for AD.

A bioluminescent and homogeneous assay for monitoring GPCR-mediated cAMP modulation and PDE activity.

3',5'-Cyclic adenosine monophosphate (cAMP), the first identified second messenger, is implicated in diverse cellular processes involving cellular metabolism, cell proliferation and differentiation, apoptosis, and gene expression. cAMP is synthesized by adenylyl cyclase (AC), which converts ATP to cAMP upon activation of Gαs-protein coupled receptors (GPCRs) in most cases and hydrolyzed by cyclic nucleotide phosphodiesterases (PDEs) to 5'-AMP. Dysregulation of cAMP signaling is implicated in a wide range of pathophysiological conditions such as cardiovascular diseases, neurodegenerative and behavioral disorders, cancers, diabetes, obesity, cataracts, and others. Therefore, cAMP targeted therapies have been and are still undergoing intense investigation for the treatment of these and other diseases. This highlights the need for developing assays to detect and monitor cAMP levels. In this study, we show cAMP Lumit assay as a highly specific homogeneous bioluminescent assay suitable for high throughput screenings with a large assay window and a wide dynamic range for cAMP detection. We believe that this assay will aid and simplify drug discovery screening efforts for cAMP signaling targeted therapies.

A Homogeneous Bioluminescent System to Monitor Cyclic Guanosine Monophosphate.

Cyclic guanosine monophosphate (cGMP) is a critical second messenger involved in various physiological processes, such as vasodilation and phototransduction. Its synthesis is stimulated by nitric oxide and natriuretic hormones, while its breakdown is mediated through highly regulated phosphodiesterase activities. cGMP metabolism has been targeted for the treatment of several diseases, including erectile dysfunction, hypertension, and heart failure. As more drugs are being sought, it will be critical to develop assays that accurately determine cGMP levels. Here, we present cGMP Lumit, a sensitive and specific bioluminescent assay to detect cGMP. We demonstrate the utility of the detection system in enzyme assays, cell-based assays, and high-throughput screening formats. It is anticipated that this assay will be of significant value to aid in further understanding the role of cGMP in physiology and support further drug discovery efforts toward the treatment of human disease.

LTP induction by structural rather than enzymatic functions of CaMKII.

Learning and memory are thought to require hippocampal long-term potentiation (LTP), and one of the few central dogmas of molecular neuroscience that has stood undisputed for more than three decades is that LTP induction requires enzymatic activity of the Ca2+/calmodulin-dependent protein kinase II (CaMKII)1-3. However, as we delineate here, the experimental evidence is surprisingly far from conclusive. All previous interventions inhibiting enzymatic CaMKII activity and LTP4-8 also interfere with structural CaMKII roles, in particular binding to the NMDA-type glutamate receptor subunit GluN2B9-14. Thus, we here characterized and utilized complementary sets of new opto-/pharmaco-genetic tools to distinguish between enzymatic and structural CaMKII functions. Several independent lines of evidence demonstrated LTP induction by a structural function of CaMKII rather than by its enzymatic activity. The sole contribution of kinase activity was autoregulation of this structural role via T286 autophosphorylation, which explains why this distinction has been elusive for decades. Directly initiating the structural function in a manner that circumvented this T286 role was sufficient to elicit robust LTP, even when enzymatic CaMKII activity was blocked.

Quantifying noise limitations of neural network segmentations in high-resolution transmission electron microscopy.

Motivated by the need for low electron dose transmission electron microscopy imaging, we report the optimal frame dose (i.e.e-/Å2) range for object detection and segmentation tasks with neural networks. The MSD-net architecture shows promising abilities over the industry standard U-net architecture in generalising to frame doses below the range included in the training set, for both simulated and experimental images. It also presents a heightened ability to learn from lower dose images. The MSD-net displays mild visibility of a Au nanoparticle at 20-30 e-/Å2, and converges at 200 e-/Å2 where a full segmentation of the nanoparticle is achieved. Between 30 and 200 e-/Å2 object detection applications are still possible. This work also highlights the importance of modelling the modulation transfer function when training with simulated images for applications on images acquired with scintillator based detectors such as the Gatan Oneview camera. A parametric form of the modulation transfer function is applied with varying ranges of parameters, and the effects on low electron dose segmentation is presented.

Stimulating ß-adrenergic receptors promotes synaptic potentiation by switching CaMKII movement from LTD to LTP mode.

Learning, memory, and cognition are thought to require synaptic plasticity, specifically including hippocampal long-term potentiation and depression (LTP and LTD). LTP versus LTD is induced by high-frequency stimulation versus low-frequency, but stimulating ß-adrenergic receptors (ßARs) enables LTP induction also by low-frequency stimulation (1 Hz) or theta frequencies (∼5 Hz) that do not cause plasticity by themselves. In contrast to high-frequency stimulation-LTP, such ßAR-LTP requires Ca2+-flux through L-type voltage-gated Ca2+-channels, not N-methyl-D-aspartate-type glutamate receptors. Surprisingly, we found that ßAR-LTP still required a nonionotropic scaffolding function of the N-methyl-D-aspartate-type glutamate receptor: the stimulus-induced binding of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) to its GluN2B subunit that mediates CaMKII movement to excitatory synapses. In hippocampal neurons, ß-adrenergic stimulation with isoproterenol (Iso) transformed LTD-type CaMKII movement to LTP-type movement, resulting in CaMKII movement to excitatory instead of inhibitory synapses. Additionally, Iso enabled induction of a major cell-biological feature of LTP in response to LTD stimuli: increased surface expression of GluA1 fused with super-ecliptic pHluorein. Like for ßAR-LTP in hippocampal slices, the Iso effects on CaMKII movement and surface expression of GluA1 fused with super-ecliptic pHluorein involved L-type Ca2+-channels and specifically required ß2-ARs. Taken together, these results indicate that Iso transforms LTD stimuli to LTP signals by switching CaMKII movement and GluN2B binding to LTP mode.

A Hybrid in Situ Approach for Cost Efficient Image Database Generation.

The visualization of results while the simulation is running is increasingly common in extreme scale computing environments. We present a novel approach for in situ generation of image databases to achieve cost savings on supercomputers. Our approach, a hybrid between traditional inline and in transit techniques, dynamically distributes visualization tasks between simulation nodes and visualization nodes, using probing as a basis to estimate rendering cost. Our hybrid design differs from previous works in that it creates opportunities to minimize idle time from four fundamental types of inefficiency: variability, limited scalability, overhead, and rightsizing. We demonstrate our results by comparing our method against both inline and in transit methods for a variety of configurations, including two simulation codes and a scaling study that goes above 19 K cores. Our findings show that our approach is superior in many configurations. As in situ visualization becomes increasingly ubiquitous, we believe our technique could lead to significant amounts of reclaimed cycles on supercomputers.

Reconstructing the exit wave of 2D materials in high-resolution transmission electron microscopy using machine learning.

Reconstruction of the exit wave function is an important route to interpreting high-resolution transmission electron microscopy (HRTEM) images. Here we demonstrate that convolutional neural networks can be used to reconstruct the exit wave from a short focal series of HRTEM images, with a fidelity comparable to conventional exit wave reconstruction. We use a fully convolutional neural network based on the U-Net architecture, and demonstrate that we can train it on simulated exit waves and simulated HRTEM images of graphene-supported molybdenum disulphide (an industrial desulfurization catalyst). We then apply the trained network to analyse experimentally obtained images from similar samples, and obtain exit waves that clearly show the atomically resolved structure of both the MoS2 nanoparticles and the graphene support. We also show that it is possible to successfully train the neural networks to reconstruct exit waves for 3400 different two-dimensional materials taken from the Computational 2D Materials Database of known and proposed two-dimensional materials.

Islet Biology During COVID-19: Progress and Perspectives.

The coronavirus-2019 (COVID-19) pandemic has had significant impact on research directions and productivity in the past 2 years. Despite these challenges, since 2020, more than 2,500 peer-reviewed articles have been published on pancreatic islet biology. These include updates on the roles of isocitrate dehydrogenase, pyruvate kinase and incretin hormones in insulin secretion, as well as the discovery of inceptor and signalling by circulating RNAs. The year 2020 also brought advancements in in vivo and in vitro models, including a new transgenic mouse for assessing beta-cell proliferation, a "pancreas-on-a-chip" to study glucose-stimulated insulin secretion and successful genetic editing of primary human islet cells. Islet biologists evaluated the functionality of stem-cell-derived islet-like cells coated with semipermeable biomaterials to prevent autoimmune attack, revealing the importance of cell maturation after transplantation. Prompted by observations that COVID-19 symptoms can worsen for people with obesity or diabetes, researchers examined how islets are directly affected by severe acute respiratory syndrome coronavirus 2. Herein, we highlight novel functional insights, technologies and therapeutic approaches that emerged between March 2020 and July 2021, written for both scientific and lay audiences. We also include a response to these advancements from patient stakeholders, to help lend a broader perspective to developments and challenges in islet research.

Using a community-based participatory research approach to meaningfully engage those with lived experience of diabetes and homelessness.

INTRODUCTION: Participatory research is a study method that engages patients in research programs, ideally from study design through to dissemination. It is not commonly used in diabetes health services research. Our objectives were to describe the process and challenges of conducting a participatory research project and to highlight the experiences of both patient co-researchers and academic researchers. RESEARCH DESIGN AND METHODS: We recruited people with lived experience of homelessness (PWLEH) and diabetes in Toronto, Canada to become patient co-researchers. They were asked to commit to attending biweekly meetings. We undertook two major research projects: concept mapping to choose a research focus; and photovoice to explore accessing healthy food while homeless. We used a convergent mixed-methods design to evaluate their experience. RESULTS: A diverse group of eight PWLEH had an average attendance of 82% over 21 meetings-despite this success, we encountered a number of challenges of conducting this research: funding, ethics approval and recruitment were particularly difficult. Group members reported that participation improved their ability to self-advocate in their diabetes care and provided them with tangible skills and social benefits. Group members stated that they valued being involved in all aspects of the research, in particular knowledge translation activities, including advocating for nutritious food at shelters; presenting to stakeholders; and meeting with policymakers. CONCLUSIONS: The use of participatory research methods enables academic researchers to support community members in pursuing research that is pertinent to them and which has a positive impact. In our study, co-researchers contributed in meaningful ways and also valued the experience.

Les défis de la gestion du diabète chez les personnes itinérantes : étude qualitative avec la méthode photovoix.

CONTEXTE: La recherche sur les défis de la prise en charge du diabète chez les personnes itinérantes qui en sont atteintes n'a pas tendance à prendre en considération le point de vue des personnes touchées. Nous avons utilisé une approche de recherche participative avec la communauté pour explorer ces défis. MÉTHODES: Nous avons recruté des cochercheurs ayant une connaissance expérientielle de l'itinérance et du diabète. Les chercheurs principaux leur ont offert une formation en recherche et ont préparé le terrain avec eux pour ce projet. Les cochercheurs ont collectivement choisi d'utiliser la méthode photovoix pour illustrer la difficulté de bien s'alimenter quand on est en situation d'itinérance et explorer en quoi cet écueil affecte plus largement la gestion du diabète. Après une formation en photographie et en éthique, les cochercheurs ont pris des photos en lien avec les objectifs du projet et rédigé des récits connexes au moyen de techniques de rédaction inspirée par des photos. Les chercheurs principaux ont analysé les photos et les récits, et ils en ont dégagé des thèmes qui se sont précisés lors de discussions de groupe. RÉSULTATS: Les 8 cochercheurs étaient atteints de diabète de type 2 (diagnostiqué de 18 mois à 23 ans auparavant) et avaient vécu en situation d'itinérance pendant des périodes allant de 8 mois à 12 ans. Nous avons dégagé 4 thèmes à partir de 17 photos et récits produits. L'itinérance affecte grandement la santé émotionnelle et mentale des personnes, ce qui nuit à leur capacité de bien gérer leur diabète. Les aliments servis dans les refuges sont rarement nutritifs ou appétissants. L'obtention d'une forme de logement peut faciliter la prise en charge du diabète en créant un environnement stable qui favorise l'autonomie, mais les coûts et le manque de connaissances sont des obstacles à la préparation de repas sains. L'itinérance complique aussi l'accès aux professionnels de la prise en charge du diabète et aux médicaments d'ordonnance. INTERPRÉTATION: Les images et les récits associés permettent de dresser un tableau frappant, complet et fidèle des défis auxquels sont confrontées les personnes en situation d'itinérance qui essaient de gérer leur diabète. Comprendre ces défis est la première étape qui permettra aux intervenants et aux décideurs de répondre aux besoins de cette population.

The impact of COVID-19 on dynamic hip screw fixation and training.

COVID-19 has had a profound impact on orthopaedic services and surgical training. AIM: This study aims to identify changes in lag screw position and Tip Apex Distance (TAD) in dynamic hip screw fixation due to changes in practice during the coronavirus pandemic and determine whether the changes resulted in improved patient outcomes and enhanced training opportunities. MATERIALS AND METHODS: Retrospective evaluation of two patient cohorts - pre-covid (n=27) and during covid (n=26) - to evaluate the TAD and lag screw position for each patient and record the grade of operating surgeon. A total of 53 patients were included. RESULTS: A mean TAD of 19.78mm and 19.80mm was calculated for cohort 1 and 2 respectively with no significant difference identified. When considering both key risk factors for lag screw cut-out (lag screw position and TAD) the number of patients with both a "satisfactory position" of the screw and a TAD <20mm, were 8/27 (29.6%) and 11/26 (42.3%), respectively. In cohort 1 3/27 cases had a consultant documented as the primary surgeon, while no consultants were documented as being present for the remaining 24 cases. 21/26 cases in cohort 2 had a consultant present some capacity, either as primary surgeon (15/21) or assistant (6/21). CONCLUSIONS: Changes in practice due to the pandemic have had some positive effects for both patients and trainees. This study has highlighted the importance of Consultant led trauma lists for improving training and surgical outcomes with a demonstrated improvement when considering both key factors associated with screw cut-out together. Ultimately some of the changes in practice should be continued in the post-covid era.

Simultaneous bilateral revision total knee arthroplasty following Abiotrophia defectiva infection.

A 65-year old man presented with 6-week history of bilateral knee pain and swelling, with difficulty mobilising. He had bilateral total knee arthroplasties in situ performed 5 years prior complicated by postoperative wound infection. Bilateral synovial fluid cultures were positive for Abiotrophia defectiva, and extensive investigations had not identified an extra-articular source of infection. Failing debridement antibiotic and implant retention procedure, the patient underwent a simultaneous bilateral 2-stage revision with articulated cement spacers impregnated with vancomycin and gentamycin. The patient received 6 weeks of intravenous antibiotics after each stage. A. defectiva is a nutritiously fastidious organism, posing a challenge for clinical laboratories to isolate and perform antimicrobial susceptibility testing, yet prosthetic joint infections caused by A. defectiva are scarce in literature and present atypically with subacute signs of chronic infection. This poses a diagnostic and therapeutic challenge, and two-stage revision is the only documented treatment that successfully eradicates the infection.

Effect of Ligand Structure on the Electron Density and Activity of Iridium Catalysts for the Borylation of Alkanes.

An in-depth study of iridium catalysts for the borylation of alkyl C-H bonds is reported. Although the borylation of aryl C-H bonds can be catalyzed by iridium complexes containing phen or bpy ligands at mild temperatures and with limiting arene, the borylation of alkyl C-H bonds remains underdeveloped. We prepared a library of phenanthrolines that contain varying substitution patterns. The corresponding phen-Ir trisboryl carbon monoxide complexes were synthesized to determine the electron-donating ability of these ligands, and the initial rates for the borylation of the C-H bonds in THF and diethoxyethane ß to oxygen catalyzed by Ir complexes containing these ligands were measured. For some subsets of these ligands, the donor ability correlated positively with the rate of C-H borylation catalyzed by the complexes containing ligands within a given subset. However, across subsets, ligands possessing similar donor properties to one another form catalysts for the borylation of alkyl C-H bonds with widely varying activity. This phenomenon was investigated computationally, and it was discovered that the stabilizing interactions between the phenanthroline ligand and the boryl ligands attached to Ir in the transition state for C-H oxidative addition could account for the differences in the activity of the catalysts that possess similar electron densities at Ir. The effect of these interactions on the borylation of secondary alkyl C-H bonds is larger than it is on the borylation of primary alkyl C-H bonds.

The brachyceran de novo gene PIP82, a phosphorylation target of aPKC, is essential for proper formation and maintenance of the rhabdomeric photoreceptor apical domain in Drosophila.

The Drosophila apical photoreceptor membrane is defined by the presence of two distinct morphological regions, the microvilli-based rhabdomere and the stalk membrane. The subdivision of the apical membrane contributes to the geometrical positioning and the stereotypical morphology of the rhabdomeres in compound eyes with open rhabdoms and neural superposition. Here we describe the characterization of the photoreceptor specific protein PIP82. We found that PIP82's subcellular localization demarcates the rhabdomeric portion of the apical membrane. We further demonstrate that PIP82 is a phosphorylation target of aPKC. PIP82 localization is modulated by phosphorylation, and in vivo, the loss of the aPKC/Crumbs complex results in an expansion of the PIP82 localization domain. The absence of PIP82 in photoreceptors leads to misshapped rhabdomeres as a result of misdirected cellular trafficking of rhabdomere proteins. Comparative analyses reveal that PIP82 originated de novo in the lineage leading to brachyceran Diptera, which is also characterized by the transition from fused to open rhabdoms. Taken together, these findings define a novel factor that delineates and maintains a specific apical membrane domain, and offers new insights into the functional organization and evolutionary history of the Drosophila retina.

A Modular and Diastereoselective 5 + 1 Cyclization Approach to N-(Hetero)Aryl Piperidines.

A new general de novo synthesis of pharmaceutically important N-(hetero)aryl piperidines is reported. This protocol uses a robustly diastereoselective reductive amination/aza-Michael reaction sequence to achieve rapid construction of complex polysubstituted ring systems starting from widely available heterocyclic amine nucleophiles and carbonyl electrophiles. Notably, the diastereoselectivity of this process is enhanced by the presence of water, and DFT calculations support a stereochemical model involving a facially selective protonation of a water-coordinated enol intermediate.

Unique osmoregulatory morphology in primitive sharks: an intermediate state between holocephalan and derived shark secretory morphology.

Discovery of an unusual rectal gland in the Atlantic sixgill shark Hexanchus vitulus led us to examine the rectal glands of 31 species of sharks to study diversity in rectal-gland morphology. Twenty-four of 31 species of sharks had digitiform glands (mean width-length ratio ± SD = 0.17 ± 0.04) previously assumed to be characteristic of all elasmobranchs regardless of habitat depth or phylogenetic age. Rectal glands from the family Somniosidae were kidney bean-shaped (mean width: length ± SD = 0.46 ± 0.05); whereas those from families Echinorhinidae and Hexanchidae were lobulate (mean width: length ± SD = 0.55 ± 0.06). Rectal gland width: length were different among species with digitiform morphology and lobulate morphology (ANOVA; R2 = 0.9; df = 15, 386; 401, F = 219.24; P < 0.001). Histological and morphological characteristics of the digitiform morphology from deep-sea sharks were similar to those from shallow-water sharks. Histology of lobulate rectal glands from hexanchids were characterised by tubule bundles separated by smooth muscle around a central lumen. Additionally, we examined plasma chemistry of four species of sharks with digitiform rectal glands and two species with lobulate rectal-gland morphology to see if there were differences between morphologies. Plasma chemistry analysis showed that urea and trimethylamine N-oxide (TMAO) followed the piezolyte hypothesis, with TMAO being highest and urea being lowest in deep-sea sharks. Among electrolytes, Na+ was highest in species with lobulate rectal glands. Hexanchids and echinorhinids both have lobulate rectal glands similar to those of holocephalans, despite the more than 400 million years separating these two groups. The morphological similarities between the lobulate rectal-gland anatomy of primitive sharks and the secretory morphology of holocephalans may represent an intermediate state between Holocephali and derived shark species.