Individual Strands Research Articles

Quantifying absolute addressability in DNA origami with molecular resolution

Self-assembled DNA nanostructures feature an unprecedented addressability with sub-nanometer precision and accuracy. This addressability relies on the ability to attach functional entities to single DNA strands in these structures. The efficiency of this attachment depends on two factors: incorporation of the strand of interest and accessibility of this strand for downstream modification. Here we use DNA-PAINT super-resolution microscopy to quantify both incorporation and accessibility of all individual strands in DNA origami with molecular resolution. We find that strand incorporation strongly correlates with the position in the structure, ranging from a minimum of 48% on the edges to a maximum of 95% in the center. Our method offers a direct feedback for the rational refinement of the design and assembly process of DNA nanostructures and provides a long sought-after quantitative explanation for efficiencies of DNA-based nanomachines.

Micro-segmental hair analysis for proving drug-facilitated crimes: Evidence that a victim ingested a sleeping aid, diphenhydramine, on a specific day

Sleeping aids are often abused in the commission of drug-facilitated crimes. Generally, there is little evidence that a victim ingested a spiked drink unknowingly because the unconscious victim cannot report the situation to the police immediately after the crime occurred. Although conventional segmental hair analysis can estimate the number of months since a targeted drug was ingested, this analysis cannot determine the specific day of ingestion. We recently developed a method of micro-segmental hair analysis using internal temporal markers (ITMs) to estimate the day of drug ingestion. This method was based on volunteer ingestion of ITMs to determine a timescale within individual hair strands, by segmenting a single hair strand at 0.4-mm intervals, corresponding to daily hair growth. This study assessed the ability of this method to estimate the day of ingestion of an over-the-counter sleeping aid, diphenhydramine, which can be easily abused. To model ingestion of a diphenhydramine-spiked drink unknowingly, each subject ingested a dose of diphenhydramine, followed by ingestion of two doses of the ITM, chlorpheniramine, 14days apart. Several hair strands were collected from each subject’s scalp several weeks after the second ITM ingestion. Diphenhydramine and ITM were detected at specific regions within individual hair strands. The day of diphenhydramine ingestion was estimated from the distances between the regions and the days of ITM ingestion. The error between estimated and actual ingestion day ranged from −0.1 to 1.9days regardless of subjects and hair collection times. The total time required for micro-segmental analysis of 96 hair segments (hair length: 3.84cm) was approximately 2days and the cost was almost the same as in general drug analysis. This procedure may be applicable to the investigation of crimes facilitated by various drugs.

Analysis of The Nucleotide Sequence Diversity of the Lassa Virus and Augmenting its Phylogenetic Tree

Lassa Virus (LASV) is the etiological catalyst for Lassa fever, an acute hemorrhagic disease with a mortality rate of 15%. Many aspects of the Lassa virus are not understood, like the causation of deafness in ⅓ of surviving patients or why symptoms are benign for 80% of those infected with the virus. Ambiguities like these suggest that there might exist some genomic heterogeneity among infecting viruses and demonstrate a need to quantify and analyze polymorphisms within LASV. Patterns that emerge from phylogenetic trees can be used to assess the structure of a population while also providing insights to the genetic makeup. The purpose of this investigation was to develop a more streamlined means of calculating nucleotide diversity within a subpopulation of Lassa virus strains and to augment a phylogenetic tree of the Lassa Virus glycoprotein precursor (GPC) segment. A total of 25 partial and complete data sequences of LASV strains were obtained from the Genbank Archives. During phase one of this investigation, the sequence data was inputted into MEGA analytical software and the sequence diversity was derived on a nucleotide level. Data from the individual strand sequences was used to augment a phylogenetic tree using Treeview X software. In phase two of this investigation, an algorithm was created using RStudio, with BSGenome and BioStrings extensions. The sequence diversity derived from the statistical analyses on MEGA was compared to that of the algorithm created. A p-value of 0.08 was found, which deviates from the accepted range of non-medical p-value of 0.00 to 0.05. It is suggested that future research focuses on creating a refurbished version of the algorithm to calculate a nucleotide diversity within a percent error of 5%.

It's good to unwind: how Hel308/HelQ helicases are good for health

In every living system the information for maintaining and propagating life is contained within a genome. This ‘book of life’ is written in the language of DNA. For life to exist, access to this is essential in order to replicate and repair genetic information for the benefit of future generations. An important group of molecular motor proteins, the helicases, allows access to this book of life by opening up the DNA double helix and exposing the DNA text of individual strands for replication, transcription, translation and repair. Like all molecular motors, helicases use chemical energy derived from the binding and hydrolysis of nucleotide triphosphates, usually ATP, to carry out mechanical tasks. They come in a variety of structures and employ different mechanisms to carry out diverse mechanical tasks. One large group of helicases are required for repairing DNA that has become damaged by radiation, chemicals or other proteins stuck on the DNA (‘roadblocks’). These DNA repair helicases are also diverse in form and function, having evolved in all forms of life to deal with many different types of DNA damage. However, they have the common property of being able to slide along and unwind DNA molecules to allow DNA repair enzymes access to fix the damage.

Initiation, Elongation, and Termination of Bacterial Cellulose Synthesis.

Cellulose is the major component of the plant cell wall and composed of β-linked glucose units. Use of cellulose is greatly impacted by its physical properties, which are dominated by the number of individual cellulose strand within each fiber and the average length of each strand. Our work described herein provides a complete mechanism for cellulose synthase accounting for its processivity and mechanism of initiation. Using ionic liquids and gel permeation chromatography, we obtain kinetic constants for initiation, elongation, and termination (release of the cellulose strand from the enzyme) for two bacterial cellulose synthases (Gluconacetobacter hansenii and Rhodobacter sphaeroides). Our results show that initiation of synthesis is primer-independent. After initiation, the enzyme undergoes multiple cycles of elongation until the strand is released. The rate of elongation is much faster than that of steady-state turnover. Elongation requires cyclic addition of glucose (from uridine diphosphate-glucose) and then strand translocation by one glucose unit. Translocations greater than one glucose unit result in termination requiring reinitiation. The rate of the strand release, relative to the rate of elongation, determines the processivity of the enzyme. This mechanism and the measured rate constants were supported by kinetic simulation. With the experimentally determined rate constants, we are able to simulate steady-state kinetics and mimic the size distribution of the product. Thus, our results provide for the first time a mechanism for cellulose synthase that accounts for initiation, elongation, and termination.

Hydrogel droplet single-cell processing: DNA purification, handling, release, and on-chip linearization.

The preparation and handling of mammalian single-cell genomic DNA is limited by the complexity bottleneck inherent to performing multi-step, multi-reagent operations in a microfluidic environment. We have developed a method for benchtop preparation of high-molecular weight, intact, single-cell genomes and demonstrate the extraction of long nucleic acid molecules in a microfluidic system. Lymphoblasts are encapsulated inside of alginate microparticles using a droplet microfluidics, and cells are lysed in bulk. The purified genomes are then delivered to and imaged on a dedicated microfluidic device. High-molecular weight DNA is protected from shear and retains its original cellular identity. Using this encapsulation protocol, we were able to extract individual nucleic acid strands on the millimeter scale inside of a microfluidic channel.

Characteristic comparison of wound and wrapped composite tubes made from carbon prepreg using modal analysis

The lightweight constructions are nowadays often based on composite materials. The best benefits are possible to obtain especially with a dispersion in the form of long fibres. In this study, the main properties of two almost identical composite rods manufactured from preimpregnated carbon fibres using two different methods will be compared. The first one is called “wrapping” which is helical layering of one wide tape in each ply. The main benefit of this method is good quality of final parts, but we are limited only by straight shapes. The second way “winding”, it is simultaneous deposition of several thinner fiber filaments. This method could handle the problem of curved shapes and fluently variable cross-sections. The disadvantage of this method is imperfect alignment of the placed fibers. This could be caused by a significant tortuosity and stickiness of the individual fiber strands. The goal of the carried work was to experimentally compare the vibrational transfer of these two almost identical rods with the same structure of layers, weight and curing parameters. Based on the certified properties of the base fibres and known boundary conditions, the material model and simulation of the vibrational response was created. The results of the empirical model compared with the experimental on the wrapped part were in good agreement. However, the use of this model also for the wound tubes is questionable, because of the found differences in natural frequencies and a shape of the transfer function. As part of the analytical study also the sensitivity analysis on the mesh quality has been done. As the conclusion is possible to say, that for determination of the main modes the mesh in a solved model could be just standard – it means without visible defects. Changes in the results achieved by improving the mesh quality are just really small. The more significant problem is that with using the contemporary knowledge and methods, it is not possible to simply create analytical model of the wound parts. The only option is trying to adjust the mechanical properties of base materials according to the empirical results.

The effect of simplifications of a numerical mesh on the results of electromagnetic analysis of the Nuclotron-type cable

The model of a single Nuclotron-type cable is presented. The goal of this model is to assess the behaviour of the cable under different loads. Two meshes with different simplifications are applied. In the first case, the superconductor in the cable is modelled as single region. Second mesh considers individual strands of the cable. The significant differences between the distributions of the electric current density obtained with both models are observed. The magnetic field remains roughly similar.

The Monitoring of selected sources in the administration of the Forests of Brno

The article concerns the monitoring of selected sources (monitored a total of 10 springs, presented the results of 5 springs) administered by the Forests of the city of Brno in the northern district of Brno-county, describing their current state, including the evaluation of selected water quality parameters. The basis of the study was to measure basic water quality parameters (water temperature, pH, oxygen and measuring the rate of flow of water in springs) and sampling for laboratory analysis. The values obtained were tabulated and graphically evaluated and compared with values in accordance with Decree no. 252/2004 Coll., which lay down the hygienic requirements for drinking water. Based on the measured values charts and graphs were compiled, and the water quality of the individual strands were assessed. The overall condition of wells and their surroundings is described in detail and assessed.

The role of hydrophobic modification on hyaluronic acid dynamics and self-assembly

The advent of nanomedicine has rejuvenated the need for increased understanding of the fundamental physicochemical properties of polymeric amphiphiles. Hyaluronic acid (HA) is a hydrophilic polysaccharide that is frequently conjugated to hydrophobic moieties and then used to entrap dyes and therapeutics. Here, we develop computational models to examine the effects of the hydrophobic modification on supramolecular behavior among three systematically designed HA derivatives substituted with alkyl chains of increasing length. Our simulations coalesce with experimentally obtained results to demonstrate the dependence of supramolecular behavior on intramolecular forces. We show that the formation of clearly defined hydrophobic domains in samples of octadecylamine-modified HA compared to HA conjugates with shorter alkyl chains is a result of more favorable hydrophobic interactions. Trends in hydrodynamic radius and polydispersity are observed in experimental results that coalesce with theoretical calculations, suggesting that supramolecular properties are dependent on the physicochemical characteristics of individual polymer strands.

Bis(dipyrrinato)zinc(II) Complex Chiroptical Wires: Exfoliation into Single Strands and Intensification of Circularly Polarized Luminescence.

One-dimensional (1D) coordination polymers (CPs) experiences limitations in exfoliation into individual strands, which hamper their utility as functional 1D nanomaterials. Here we synthesize chiral 1D-CPs that feature the bis(dipyrrinato)zinc(II) complex motif. They can be exfoliated into single strands upon sonication in organic media, retaining lengths of up to 3.19 μm (ca. 2600 monomer units). Their chiroptical structure allows the exfoliated wires to show circularly polarized luminescence at an intensity 5.9 times that of reference monomer complexes.

Unique growth strategy in the Earth’s first trees revealed in silicified fossil trunks from China

Cladoxylopsida included the earliest large trees that formed critical components of globally transformative pioneering forest ecosystems in the Mid- and early Late Devonian (ca. 393-372 Ma). Well-known cladoxylopsid fossils include the up to ∼1-m-diameter sandstone casts known as Eospermatopteris from Middle Devonian strata of New York State. Cladoxylopsid trunk structure comprised a more-or-less distinct cylinder of numerous separate cauline xylem strands connected internally with a network of medullary xylem strands and, near the base, externally with downward-growing roots, all embedded within parenchyma. However, the means by which this complex vascular system was able to grow to a large diameter is unknown. We demonstrate-based on exceptional, up to ∼70-cm-diameter silicified fossil trunks with extensive preservation of cellular anatomy from the early Late Devonian (Frasnian, ca. 374 Ma) of Xinjiang, China-that trunk expansion is associated with a cylindrical zone of diffuse secondary growth within ground and cortical parenchyma and with production of a large amount of wood containing both rays and growth increments concentrically around individual xylem strands by normal cambia. The xylem system accommodates expansion by tearing of individual strand interconnections during secondary development. This mode of growth seems indeterminate, capable of producing trees of large size and, despite some unique features, invites comparison with secondary development in some living monocots. Understanding the structure and growth of cladoxylopsids informs analysis of canopy competition within early forests with the potential to drive global processes.

Different localizations of drugs simultaneously administered in a strand of hair by micro‐segmental analysis

Segmental hair analysis is used to estimate the time of drug intake at monthly precision in drug-related crimes. Previously, we advanced this analytical method to specify the day of drug intake by cutting a strand of hair into 0.4-mm segments, which correspond to daily hair growth. Herein, we investigated the distributions of 7 compounds in a strand of hair using micro-segmental analysis. Several strands of hair were collected 33.1-229.4 days after subjects were administered 4 pharmaceutical products that contained 10 drugs in single doses within 32 hours. The administered drugs and resulting metabolites were extracted from 0.4-mm hair segments and quantified using liquid chromatography-tandem mass spectrometry. Acidic and neutral compounds were detected at low amounts in any of the hair segments analyzed. Epinastine, fexofenadine, dihydrocodeine, chlorpheniramine, and the chlorpheniramine metabolite, desmethylchlorpheniramine each was localized to 2 regions within a strand of hair. By contrast, methylephedrine and its metabolite, ephedrine, each was localized to only a region. Among 20 individual strands of hair associated with different subjects and head regions, few differences in the shapes of drug concentration-hair segment curves for each compound were detected. Our data indicated that 2 mechanisms for drug uptake into hair can operate depending on drug properties and that co-administered drugs can be localized to different regions in a strand of hair. Micro-segmental analysis may aid in the identification of the day of drug intake and help to elucidate the mechanisms of drug uptake into hair.

Physical Modelling of Tundish Slag Entrainment under Various Technological Conditions

AbstractThis paper deals with the issue of physical modelling of vortexes creation and tundish slag entrainment over the mouth of the nozzle into the individual casting strands. Proper physical model is equivalent to the operational continuous casting machine No. 2 in TŘINECKÉ ŽELEZÁRNY, a.s. Physical modelling methodology and simulated operational conditions are shortly described. Physical modelling was used for the evaluation of current conditions of steel casting at the application of different impact pads in the tundish. Further, laboratory measurement on the physical model aiming the determination of exact critical periods of vortexes creation and study of the slag entrainment as a consequence of changes in surface level during the tundish refilling to standard level were realised. The obtained results were analysed and discussed.

Microhaplotypes for ancestry prediction

Microhaplotypes (MHs) are loci of two or more SNPs within a short distance from each other (<300 nucleotides) with three or more allelic combinations. Massively parallel sequencing (MPS) is clonal sequencing of individual strands, thus it can distinguish the parental haplotypes at a locus. MH alleles within a locus all have the same size, do not generate stutter fragments, and have lower mutation rates than STRs. The goal of this project was to determine whether MHs could provide biogeographic ancestry prediction.A total of 278 samples including 76 African-Americans (AAs), 110 European-Americans (EAs) and 92 South West Hispanics (SWHISPs) were selected and genotyped using a 74-plex assay on the Ion Chef™ and Ion S5™ MPS platform. Results were used to generate allele frequencies for the three populations. A set of test samples (28) was genotyped and used to calculate the random match probability (RMP) in the three populations. Additionally, PHASE inferred allele frequencies from 24 populations obtained from ALFRED were used to calculate the RMP of the test samples in each population. A mixture study was also performed to evaluate the detection limit for the minor contributor of the MH 74-plex.Among the populations studied, the RMP averaged remarkably higher in each individual’s self-identified population of “origin”. For example, AAs had the highest RMP in African and African-American populations. In the mixture study results showed that alleles from the minor contributor were detectable at an 80:1 major/minor ratio when the input DNA was 10ng and 40:1 when the input amount was 1ng with a RMP equal to or greater than a typical 15 loci STR profile demonstrating that MHs can be an effective tool for ancestry prediction and mixture analysis.

Dynamic properties of unbonded, multi-strand beams subjected to flexural loading

Abstract Beam-like structures, constructed from many long strands that are constrained rather than bonded together, can provide appreciable levels of structural damping through friction between individual strands. This paper describes experimental and numerical studies, carried out on square-section metal beams, which are aimed at improving understanding of the relationship between construction and performance. A beam is formed from a pack of square-section strands that is held together at various compression loads with pre-calibrated clamps. Flexural deformation of the assembled beam is simulated using standard finite element analysis employing simple Coulomb friction at the interfaces. The validity of the assumptions used in the models is confirmed by comparison with three point bend tests on a regular nine strand construction at several different clamp loads. Dynamic loss factors for this beam are obtained by conducting forced vibration tests, which show that the damping is insensitive to frequency. Subsequent numerical studies are used to investigate the effects of increasing the number of strands whilst maintaining the overall cross-section geometry of the beam. It is found that the system stiffness drops and loss factor increases when more strands are used for a maintained beam cross-section. Interestingly, the energy dissipated by each beam construction is almost the same. These results provide a vital and necessary insight into the physics for stranded structures and materials that are largely prevalent in mechanical (e.g. cables) and electrical (e.g. wires) elements.

Theoretical simulation of the infrared signature of mechanically stressed polymer solids.

Mechanical stress leads to deformation of strands in polymer solids, including elongation of covalent bonds and widening of bond angles, which changes the infrared spectrum. Here, the infrared spectrum of solid polymer samples exposed to mechanical stress is simulated by density functional theory calculations. Mechanical stress is described with the external force explicitly included (EFEI) method. The uneven distribution of the external stress on individual polymer strands is accounted for by a convolution of simulated spectra with a realistic force distribution. N-Propylpropanamide and propyl propanoate are chosen as model molecules for polyamide and polyester, respectively. The effect of a specific force on the polymer backbone is a redshift of vibrational modes involving the C–N and C–O bonds in the backbone, while the free C–O stretching mode perpendicular to the backbone is largely unaffected. The convolution with a realistic force distribution shows that the dominant effect on the strongest infrared bands is not a shift of the peak position, but rather peak broadening and a characteristic change in the relative intensities of the strongest bands, which may serve for the identification and quantification of mechanical stress in polymer solids.

ConcatSeq: A method for increasing throughput of single molecule sequencing by concatenating short DNA fragments

Single molecule sequencing (SMS) platforms enable base sequences to be read directly from individual strands of DNA in real-time. Though capable of long read lengths, SMS platforms currently suffer from low throughput compared to competing short-read sequencing technologies. Here, we present a novel strategy for sequencing library preparation, dubbed ConcatSeq, which increases the throughput of SMS platforms by generating long concatenated templates from pools of short DNA molecules. We demonstrate adaptation of this technique to two target enrichment workflows, commonly used for oncology applications, and feasibility using PacBio single molecule real-time (SMRT) technology. Our approach is capable of increasing the sequencing throughput of the PacBio RSII platform by more than five-fold, while maintaining the ability to correctly call allele frequencies of known single nucleotide variants. ConcatSeq provides a versatile new sample preparation tool for long-read sequencing technologies.

Comparison of modal characteristic of wrapped and winded composite tubes from carbon prepreg

Constructions based on long fiber composite frames gaining significant and irreplaceable position through almost all traffic sectors. The aim of this presented work is a study of composite rods made by two ways from pre impregnated fibres. In technical practice, one can find applications of so called “prepregs”, especially for so-called wrapping, which is a gradual helical layering of wide tapes. This is a quite simply method with a good quality of final parts, but the using is limited only for straight shapes. Therefore, a method called winding, which is a simultaneous deposition of larger number of fiber filaments was used to handle the problem for curved shapes and fluently changed cross-sections. Even though this method could expand the using of presaturated fibres, in a real case compared with wrapped parts there is evident imperfect alignment and mutually storing of placed fibers. This could be mainly caused by a significant tortuosity and stickiness of the individual fiber strands. The objective the carried work was to experimentally compare the vibrational - modal behavior of two almost identical rods. One was winded and the second kind was wrapped with the same structure of layers, weight and curing parameters. The results of the two compared tubes, that should be theoretically identical, were significantly different. Another step was compilation of a numerical model and verify its suitability for parts created with the two mentioned production methods. The numerical model was in a good agreement with the empirical obtained from the experiment with wrapped parts. However, the using of this standard model also for winded tube is questionable, because of the significantly differences in founded natural frequencies.

Folding Topology of a Short Coiled-Coil Peptide Structure Templated by an Oligonucleotide Triplex.

The rational design of a well-defined protein-like tertiary structure formed by small peptide building blocks is still a formidable challenge. By using peptide-oligonucleotide conjugates (POC) as building blocks, we present the self-assembly of miniature coiled-coil α-helical peptides guided by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex under physiological pH, and to be capable of templating the three peptide sequences to constitute a small coiled-coil motif displaying remarkable α-helicity. The formed trimeric complex was characterized by ultraviolet thermal denaturation, gel electrophoresis, circular dichroism (CD) spectroscopy, small-angle X-ray scattering (SAXS), and molecular modeling. Stabilizing cooperativity was observed between the trimeric peptide and the oligonucleotide triplex domains, and the overall molecular size (ca. 12 nm) in solution was revealed to be independent of concentration. The topological folding of the peptide moiety differed strongly from those of the individual POC strands and the unconjugated peptide, exclusively adopting the designed triple helical structure.