Probing the Structure and in Silico Stability of Cargo Loaded DNA Icosahedron using MD Simulations

Platonic solids such as polyhedra based on DNA have been deployed for multifarious applications such as RNAi delivery, biological targeting and bioimaging. All of these applications hinge on the capability of DNA polyhedra for molecular display with high spatial precision. Therefore high resolution structural models of such polyhedra are critical to widen their applications in both materials and biology. Here, we present an atomistic model of a well-characterized DNA icosahedron, with demonstrated versatile functionalities in biological systems. We study the structure and dynamics of this DNA icosahedron using fully atomistic molecular dynamics simulation in explicit water and ions. The major modes of internal motion have been identified using principal component analysis. We provide a quantitative estimate of the radius of gyration (Rg), solvent accessible surface area (SASA) and volume of the icosahedron which is essential to estimate its maximal cargo carrying capacity. Importantly, our simulation of gold nanoparticles (AuNP) encapsulated within DNA icosahedra revealed enhanced stability of the AuNP loaded DNA icosahedra compared to empty icosahedra. This is consistent with experimental results that show high yields of cargo-encapsulated DNA icosahedra that have led to its diverse applications for precision targeting. These studies reveal that the stabilizing interactions between the cargo and the DNA scaffold powerfully positions DNA polyhedra as targetable nanocapsules for payload delivery. These insights can be exploited for precise molecular display for diverse biological applications.Comment: 46 pages, 16 Figures and 3 Tables, Accepted for publication in Nanoscal

Structural DNA nanotechnology: from bases to bricks, from structure to function.

ABSTRACT The two fields of structural DNA nanotechnology and functional nucleic acids have been independently coevolving, with the former seeking to arrange and bring about movement of nucleic acid modules precisely and with control in space and the latter producing modules with incredible diversity in effective recognition and function. Here, we track the key developments in structural DNA nanotechnology that reveal a current trend that is seeing the integration of functional nucleic acid modules into their architectures to access a range of new functions. This contribution will seek to provide a perspective for the field of structural DNA nanotechnology where the integration of such functional modules on precisely controlled architectures can uncover phenomena of interest to physical chemists. D NA has proven to be a powerful material for construction on the nanoscale on the basis of the following properties: (i) the availability of automated synthetic methods and continually dropping costs, (ii) chemical robustness that confers stability on the resultant architectures and their subsequent ability to be functional under a variety of environmental conditions, (iii) the uniformly rodlike nature of the DNA double helix irrespective of its primary sequence, (iv) the specificity of Watson-Crick base pairing, which functions as an easily engineerable, site-specific, molecular-scale glue applicable to any DNA double helix, (v) the periodic nature of the DNA double helix and the predictable nature of sequence-specific thermal stability, both of which predispose it to computational methods to design and fabricate superarchitectures, (vi) the availability of well-characterized biochemical and molecular biological methods to cut, copy, and covalently link B-DNA double helices sequencespecifically, which allows manipulation of the construction material, (vii) the modular nature of the DNA scaffold that allows fabrication of architectures that are complex in terms of both structure and function when multiple modules are appended to each other, and (viii) single-stranded DNA sequences, called functional nucleic acids, which can fold and offer three-dimensional cavities suited to bind with great specificity a range of molecular entities with diverse function. In 1982, Ned Seeman proposed that DNA, which until then had been thought of as a linear polymer, could be used to make branched architectures by using stable artificial junctions with helical DNA limbs radiating from a central node. 1 These structures were analogous to metastable naturally occurring DNA motifs, such as the replication fork and Holliday junction. "It appears to be possible to generate covalently joined...networks of nucleic acids which are periodic in connectivity and perhaps in space." 1 This marked the origin of structural DNA nanotechnology that seeks to create defined architectures on the nanoscale using sequences of DNA that self-assemble into rigid rods that are, in turn, connected to form superarchitectures of precise dimensions. In 1999, it was shown that DNA could switch between two forms (the B-form and the Z-form), and this motion could be transduced along a DNA architecture, making it undergo a twisting motion. 2 Thus began a complementary aspect of structural DNA nanotechnology, of bringing about defined molecular-scale movements of DNA architectures triggered by the addition of input stimuli that are chemical, photonic, thermal, or electrical in nature. Functional nucleic acids are obtained from a test tube evolution method called SELEX independently conceptualized by the Szostak and Gold groups. 3,4 It uses molecular biology tools to pick out from a library of ∼10 15 different DNA (or RNA) sequences, a subset of sequences based on a given selection criterion and amplify them. 5 When subjected to the same selection criterion repeatedly with progressively higher stringencies, it is possible to progressively enrich from the library, a pool of DNA (or RNA) sequences with a specific functionality. If the selection criterion is the recognition of a target molecule, then selected single-stranded DNA (ssDNA) sequences are capable of binding to the target with high specificity and affinity. Thus, SELEX has yielded DNA sequences that can bind a huge variety of chemical entities ranging from small molecules to proteins, peptides, transition-stat

Friction Mediates Scission of Tubular Membranes Scaffolded by BAR Proteins

International audienceMembrane scission is essential for intracellular trafficking. While BAR domain proteins such as endophilin have been reported in dynamin-independent scission of tubular membrane necks, the cutting mechanism has yet to be deciphered. Here, we combine a theoretical model, in vitro, and in vivo experiments revealing how protein scaffolds may cut tubular membranes. We demonstrate that the protein scaffold bound to the underlying tube creates a frictional barrier for lipid diffusion; tube elongation thus builds local membrane tension until the membrane undergoes scission through lysis. We call this mechanism friction-driven scission (FDS). In cells, motors pull tubes, particularly during endocytosis. Through reconstitution, we show that motors not only can pull out and extend protein-scaffolded tubes but also can cut them by FDS. FDS is generic, operating even in the absence of amphipathic helices in the BAR domain, and could in principle apply to any high-friction protein and membrane assembly

Comparison of Small Gut and Whole Gut Microbiota of First-Degree Relatives With Adult Celiac Disease Patients and Controls

Recent studies on celiac disease (CeD) have reported alterations in the gut microbiome. Whether this alteration in the microbial community is the cause or effect of the disease is not well understood, especially in adult onset of disease. The first-degree relatives (FDRs) of CeD patients may provide an opportunity to study gut microbiome in pre-disease state as FDRs are genetically susceptible to CeD. By using 16S rRNA gene sequencing, we observed that ecosystem level diversity measures were not significantly different between the disease condition (CeD), pre-disease (FDR) and control subjects. However, differences were observed at the level of amplicon sequence variant (ASV), suggesting alterations in specific ASVs between pre-disease and diseased condition. Duodenal biopsies showed higher differences in ASVs compared to fecal samples indicating larger disruption of the microbiota at the disease site. The duodenal microbiota of FDR was characterized by significant abundance of ASVs belonging to Parvimonas, Granulicatella, Gemella, Bifidobacterium, Anaerostipes, and Actinomyces genera. The duodenal microbiota of CeD was characterized by higher abundance of ASVs from genera Megasphaera and Helicobacter compared to the FDR microbiota. The CeD and FDR fecal microbiota had reduced abundance of ASVs classified as Akkermansia and Dorea when compared to control group microbiota. In addition, predicted functional metagenome showed reduced ability of gluten degradation by CeD fecal microbiota in comparison to FDRs and controls. The findings of the present study demonstrate differences in ASVs and predicts reduced ability of CeD fecal microbiota to degrade gluten compared to the FDR fecal microbiota. Further research is required to investigate the strain level and active functional profiles of FDR and CeD microbiota to better understand the role of gut microbiome in pathophysiology of CeD

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

SummaryBackground Azithromycin has been proposed as a treatment for COVID-19 on the basis of its immunomodulatoryactions. We aimed to evaluate the safety and efficacy of azithromycin in patients admitted to hospital with COVID-19.Methods In this randomised, controlled, open-label, adaptive platform trial (Randomised Evaluation of COVID-19Therapy [RECOVERY]), several possible treatments were compared with usual care in patients admitted to hospitalwith COVID-19 in the UK. The trial is underway at 176 hospitals in the UK. Eligible and consenting patients wererandomly allocated to either usual standard of care alone or usual standard of care plus azithromycin 500 mg once perday by mouth or intravenously for 10 days or until discharge (or allocation to one of the other RECOVERY treatmentgroups). Patients were assigned via web-based simple (unstratified) randomisation with allocation concealment andwere twice as likely to be randomly assigned to usual care than to any of the active treatment groups. Participants andlocal study staff were not masked to the allocated treatment, but all others involved in the trial were masked to theoutcome data during the trial. The primary outcome was 28-day all-cause mortality, assessed in the intention-to-treatpopulation. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936.Findings Between April 7 and Nov 27, 2020, of 16 442 patients enrolled in the RECOVERY trial, 9433 (57%) wereeligible and 7763 were included in the assessment of azithromycin. The mean age of these study participants was65·3 years (SD 15·7) and approximately a third were women (2944 [38%] of 7763). 2582 patients were randomlyallocated to receive azithromycin and 5181 patients were randomly allocated to usual care alone. Overall,561 (22%) patients allocated to azithromycin and 1162 (22%) patients allocated to usual care died within 28 days(rate ratio 0·97, 95% CI 0·87–1·07; p=0·50). No significant difference was seen in duration of hospital stay (median10 days [IQR 5 to >28] vs 11 days [5 to >28]) or the proportion of patients discharged from hospital alive within 28 days(rate ratio 1·04, 95% CI 0·98–1·10; p=0·19). Among those not on invasive mechanical ventilation at baseline, nosignificant difference was seen in the proportion meeting the composite endpoint of invasive mechanical ventilationor death (risk ratio 0·95, 95% CI 0·87–1·03; p=0·24).Interpretation In patients admitted to hospital with COVID-19, azithromycin did not improve survival or otherprespecified clinical outcomes. Azithromycin use in patients admitted to hospital with COVID-19 should be restrictedto patients in whom there is a clear antimicrobial indication

Studies on the copolymerization of cyclic ketene acetals with styrene

548-557   The paper describes the synthesis, characterization and copolymerization behaviour of 2-ethylidene-4-methyl-1, 3-dioxolane (A)/2-ethylidene-4 ethyl-1,3-dioxolane (B)/2-ethylidene-1,3-dioxepane (C)/2-ethylidene-1,3-dioxane (D) monomers with styrene. Homopolymerization and copolymerization of these monomers with styrene was carried out in bulk using free radical initiator. Structural characterization was done using FTIR and 1H-NMR spectroscopy. 1H NMR and FTIR spectroscopy was also used for the calculation of percentage ring opening and copolymer composition. The ring opening in case of ethylidene monomers having seven membered ring was maximum and minimum in case of ethylidene monomers having five membered ring.    Thermal characterization was done using differential scanning calorimetry and thermogravimetry in nitrogen atmosphere. Homopolymers of monomers A, C and D had Tg values of 50,-60 and -62oC respectively. Tg of the copolymers decreased with increasing amounts of cyclic ketene acetal monomers in the copolymers. All the homopolymers and copolymers were stable upto 350°C. Biodegradability of the polymers was also evaluated using the enzyme lipase. Incorporation of 0.2 mole fraction of these cyclic ketene acetals in polystyrene backbone improves its biodegradability

The roles of dynein and myosin VI motor proteins in endocytosis

International audienceABSTRACT Endocytosis is indispensable for multiple cellular processes, including signalling, cell adhesion, migration, as well as the turnover of plasma membrane lipids and proteins. The dynamic interplay and regulation of different endocytic entry routes requires multiple cytoskeletal elements, especially motor proteins that bind to membranes and transport vesicles along the actin and microtubule cytoskeletons. Dynein and kinesin motor proteins transport vesicles along microtubules, whereas myosins drive vesicles along actin filaments. Here, we present a brief overview of multiple endocytic pathways and our current understanding of the involvement of these motor proteins in the regulation of the different cellular entry routes. We particularly focus on structural and mechanistic details of the retrograde motor proteins dynein and myosin VI (also known as MYO6), along with their adaptors, which have important roles in the early events of endocytosis. We conclude by highlighting the key challenges in elucidating the involvement of motor proteins in endocytosis and intracellular membrane trafficking