Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles.

The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelles, the sites of replication of viral genomic RNA (vgRNA). To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain numerous vgRNA molecules along with the replication enzymes and clusters of viral double-stranded RNA (dsRNA). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of endoplasmic reticulum (ER) markers and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are encapsulated into DMVs, which have membranes derived from the host ER. These organelles merge into larger vesicle packets as infection advances. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles.

The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelle where the replication of viral genomic RNA (vgRNA) occurs. To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain vgRNA clusters along with viral double-stranded RNA (dsRNA) clusters and the replication enzyme, encapsulated by membranes derived from the host endoplasmic reticulum (ER). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of ER labels and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are enclosed by DMVs at early infection stages which then merge into vesicle packets as infection progresses. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

Angle distortion model for predicting enediyne activation towards Bergman cyclization: an alternate to the distance theory.

The kinetics of Bergman cyclization (BC) of enediynes into 1,4-benzene diradicals (also known as p-benzynes) have attracted interest ever since the discovery of natural enediynes which pointed out a surprising reactivity profile difference across enediynes with varying structural architectures. From the analysis of experimental kinetic data, several models were proposed to have a structure-kinetics correlation, out of which, the cd-distance model and the transition state model are the most accepted ones. Recently, Houk et al. introduced a distortion model to explain the regioselectivity of nucleophilic addition to unsymmetrical o-benzynes based on the geometry of the transition state. In the case of BC, since the reaction is endothermic, the transition state geometrically resembles the product structure which implies that in the reaction pathway, the sp-carbons of enediynes are transformed into the trigonal sp2 carbons of the benzenoid product. Thus, greater bending of the interior angles at the proximal alkyne carbons in the enediynes will lead to a lower activation barrier for the BC and hence faster cyclization. This hypothesis has been tested on a series of enediynes including natural product surrogates and the extent of deviation correlates well with the kinetic results. A cut-off value for the average internal proximal angles has been proposed to categorize enediynes as per their reactivity under ambient conditions. We believe that this distortion theory offers an alternative model in designing new unnatural enediynes with desired kinetic stabilities.

PINE: Post-Quantum Based Incentive Technique for Non-Cooperating Nodes in Internet of Everything.

The Internet of Everything (IoE) is a smart system that interconnects smart entities by incorporating low-cost or low-energy gadgets that are useful for communication with people, processes, data, and devices/things. In such an instantaneously connected environment, network-enabled heterogeneous devices may exhibit non-cooperative behaviour which may lead to the degradation of the network. To address this performance degradation, the proposed Post-quantum based Incentive technique for Non-cooperating nodes in internet of Everything (PINE) protocol provides an end-to-end reliable solution by incorporating location-aware post-quantum encryption in these networks while addressing the non-cooperative behaviour of the nodes by employing an effective strategy in a bi-directional multi-hop relay environment. This proposed protocol further aims to evaluate the consequences of non-cooperative nodes by considering various metrics, namely, number of nodes, message size, execution time, memory consumption, average residual energy, percentage of selfish nodes, and blackhole nodes detection, aiming to achieve significant accuracy in an IoE environment.

Evaluation of the feasibility and acceptability of ReWin-A digital therapeutic rehabilitation innovation for people with stroke-related disabilities in India.

Background: Developing culturally appropriate, scalable interventions to meet the growing needs for stroke rehabilitation is a significant problem of public health concern. Therefore, systematic development and evaluation of a scalable, inclusive, technology-driven solution for community-based stroke care are of immense public health importance in India. ReWin is a digital therapeutics platform that was developed systematically. This study aimed to evaluate its feasibility and acceptability in an Indian context. Objectives: Phase-1: To pilot the intervention for identifying operational issues and finalize the intervention. Phase-2: To assess the feasibility and acceptability of ReWin intervention in an Indian context. Methods: Design: Mixed-methods research design. Setting: Participant's home and rehabilitation centers. Participants were selected from rehabilitation centers in South India. Participants: Ten stroke survivors and their caregivers, as well as four rehabilitation service providers were recruited for phase 1. Thirty stroke survivors who were treated and discharged from the hospital, and their caregivers as well as 10 rehabilitation service providers were recruited for Phase 2. Intervention: ReWin a digital therapeutic platform with the provider and patient app for the rehabilitation of physical disabilities following stroke was piloted. Process: Evaluation of the intervention was completed in two phases. In the first phase, the preliminary intervention was field-tested with 10 stroke survivors and four rehabilitation service providers for 2 weeks. In the second phase, the finalized intervention was provided to a further 30 stroke survivors to be used in their homes with support from their carers as well as to 10 rehabilitation service providers for 4 weeks. Outcome measures: Primary outcomes: (1) operational difficulties in using the ReWin intervention; (2) feasibility and acceptability of the ReWin intervention in an Indian setting. Results: Field-testing identified operational difficulties related to 1. Therapeutic content; 2. Format; 3. Navigation; 4. Connectivity, 5. Video-streaming, 6. Language; and 7. Comprehensibility of the animated content. The intervention was reviewed, revised and finalized before pilot testing. Findings from the pilot testing showed that the ReWin intervention was feasible and acceptable. About 76% of the participants had used ReWin for more than half of the intervention period of 4 weeks. Ninety percentage of the stroke care providers and about 60% of the stroke survivors and caregivers felt that the content of ReWin was very relevant to the needs of the stroke survivors. Forty percentage of the stroke survivors and caregivers rated ReWin intervention as excellent. Another 45% of the stroke survivors and caregivers as well as 90% of the stroke care providers rated ReWin intervention as very good based on its overall credibility, usability, and user-friendliness. Conclusions: ReWin has all the essential components to connect care providers and consumers not just for stroke rehabilitation but for several other health conditions with the use of several other technological features that support rehabilitation of persons with disabilities and strengthen rehabilitation in health systems worldwide. It is critical to amalgamate ReWin and other evidence-based interventions for rehabilitation to innovate scalable solutions and promote universal health coverage for stroke care worldwide.

Protamine loops DNA in multiple steps.

Protamine proteins dramatically condense DNA in sperm to almost crystalline packing levels. Here, we measure the first step in the in vitro pathway, the folding of DNA into a single loop. Current models for DNA loop formation are one-step, all-or-nothing models with a looped state and an unlooped state. However, when we use a Tethered Particle Motion (TPM) assay to measure the dynamic, real-time looping of DNA by protamine, we observe the presence of multiple folded states that are long-lived (∼100 s) and reversible. In addition, we measure folding on DNA molecules that are too short to form loops. This suggests that protamine is using a multi-step process to loop the DNA rather than a one-step process. To visualize the DNA structures, we used an Atomic Force Microscopy (AFM) assay. We see that some folded DNA molecules are loops with a ∼10-nm radius and some of the folded molecules are partial loops-c-shapes or s-shapes-that have a radius of curvature of ∼10 nm. Further analysis of these structures suggest that protamine is bending the DNA to achieve this curvature rather than increasing the flexibility of the DNA. We therefore conclude that protamine loops DNA in multiple steps, bending it into a loop.

Improved Electrostatic Embedding for Fragment-Based Chemical Shift Calculations in Molecular Crystals.

Fragment-based methods predict nuclear magnetic resonance (NMR) chemical shielding tensors in molecular crystals with high accuracy and computational efficiency. Such methods typically employ electrostatic embedding to mimic the crystalline environment, and the quality of the results can be sensitive to the embedding treatment. To improve the quality of this embedding environment for fragment-based molecular crystal property calculations, we borrow ideas from the embedded ion method to incorporate self-consistently polarized Madelung field effects. The self-consistent reproduction of the Madelung potential (SCRMP) model developed here constructs an array of point charges that incorporates self-consistent lattice polarization and which reproduces the Madelung potential at all atomic sites involved in the quantum mechanical region of the system. The performance of fragment- and cluster-based 1H, 13C, 14N, and 17O chemical shift predictions using SCRMP and density functionals like PBE and PBE0 are assessed. The improved embedding model results in substantial improvements in the predicted 17O chemical shifts and modest improvements in the 15N ones. Finally, the performance of the model is demonstrated by examining the assignment of the two oxygen chemical shifts in the challenging γ-polymorph of glycine. Overall, the SCRMP-embedded NMR chemical shift predictions are on par with or more accurate than those obtained with the widely used gauge-including projector augmented wave (GIPAW) model.